Targeting abcb5 for cancer therapy

ABSTRACT

The invention related to methods for treating a subject by manipulating ABCB5 on a cell as well as related products. The methods include methods of treating cancer using ABCB5 binding molecules such as antibodies and fragments thereof.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application No. 61/007,059, filed Dec. 11, 2007, andApplication No. 60/923,128, filed Apr. 12, 2007, the entire contents ofwhich is hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH Grant No.1R01CA113796-01A 1. The Government has certain rights in this invention.

BACKGROUND OF INVENTION

Human malignant melanoma is a highly chemorefractory cancer. There arecurrently not many effective treatment options. Malignant melanoma ofthe skin is highly prevalent in the United States, with 1 in 63 men andwomen afflicted during their lifetime. Of those, 11% are diagnosed afterthe cancer has spread to regional lymph nodes or directly beyond theprimary site and 3% after the cancer has already metastasized (distantstage), with corresponding 5-year relative survival rates of 63.8% and16.0%.

SUMMARY OF INVENTION

The invention is based at least in part on the discovery thatchemoresistant ABCB5+ tumor stem cells contribute to the development ofcancers such as melanoma and that these cells can be targeted to treatthe cancer. ABCB5 targeting may be employed as either a stand-alonetherapeutic approach to disseminated disease, or as an adjunctivetherapy to sensitize cancer cells to chemotherapeutic agents, especiallyin those patients with currently refractory metastatic disease. Anadvantage of ABCB5-targeted therapeutic approaches is that they aredirected at tumorigenic stem cells, whereas conventional therapeuticstarget only the bulk population of tumor cells.

In some aspects the invention relates to a method of delivering atherapeutic agent to an intracellular compartment of a cell bycontacting a cell with an isolated molecule that selectively binds toABCB5 conjugated to a therapeutic agent in an effective amount todeliver the therapeutic agent to an intracellular compartment of thecell.

In some embodiments the isolated molecule that selectively binds toABCB5 is an isolated peptide. In other embodiments it is a smallmolecule. The isolated peptide may be, for instance, an antibody orantigen binding fragment thereof or an scFv.

The therapeutic agent may be, in some embodiments, a toxin, an siRNA, achemotherapeutic agent or a therapeutic antibody.

The method involves, in other embodiments the step of contacting a cellwith an isolated molecule that selectively binds to a surface markersuch as CD49e, CD133, CD166, BMPR1a, TIR-1, VE-cadherin (CD144) ornestin.

According to another aspect of the invention a composition is providedof an isolated peptide that selectively binds to ABCB5 and comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7 and SEQ ID NO:8, or functionally equivalent variants thereofcontaining conservative substitutions, wherein the isolated peptide isnot mAb 3C2-1D12.

In other aspects of the invention a composition of an isolated peptidethat selectively binds to ABCB5 and comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ IDNO:8, or functionally equivalent variants thereof containingconservative substitutions is provided. The isolated antibody orantibody fragment is present in an effective amount for enhancingchemosensitization in a human subject.

According to yet another aspect of the invention a composition of anisolated peptide that selectively binds to ABCB5 and comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7and SEQ ID NO:8, or functionally equivalent variants thereof containingconservative substitutions is provided. The isolated peptide ispreferably co-formulated with a therapeutic agent. The isolated peptide,in some embodiments, is conjugated to the therapeutic agent. In otherembodiments the therapeutic agent is selected from the group consistingof camptothecin 9-NH2, mitoxantrone, camptothecin 7-Cl, pyrazofurin,menogaril, camptothecin 20 ester, camptothecin, amsacrine, etopside,anthrapyrazole-derivitive, terniposide, camptothecin 11-formyl,camptothecin 10-OH, daunorubicin, doxydoxorubicin, doxorubicin,oxanthrazoole, camptothecin 11-HOMe, zorubicin, uracil mustard,piperazinedione, hepsulfam, melphalan, bisantrene, triethylenemelamine,spiromustine, Yoshi-864, chlorambucil, piperazine mustard, hydroyurea,porfiromycin, mechlorethamine, fluorodopan, mitomycin, cytarabine(araC), dianhydrogalactitol, gemcitabine, thiotepa,N,N-dibenzyl-daunomycin, teroxirone, and aphidicolin-glycinate.

A kit is provided according to other aspects of the invention. The kitincludes a container housing an isolated peptide that selectively bindsto ABCB5 and comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11 and SEQ ID NO:12, or functionally equivalentvariants thereof containing conservative substitutions, and instructionsfor administering the isolated peptide to a human subject.

A method for treating a subject is provided according to other aspectsof the invention. The method involves systemically administering anisolated peptide that selectively binds to ABCB5 and comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7and SEQ ID NO:8, or functionally equivalent variants thereof containingconservative substitutions to a subject having cancer in an effectiveamount to treat the cancer.

Method for treating a subject by administering any one of thecompositions described herein to a subject having cancer in an effectiveamount to treat the cancer is also provided. A method for treating asubject is provided according to other aspects of the invention. Themethod involves administering an isolated peptide that selectively bindsto ABCB5 and comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, or functionallyequivalent variants thereof containing conservative substitutions and achemotherapeutic agent, to a subject having cancer in an effectiveamount to treat the cancer.

According to other aspects of the invention a method is provided fortreating a subject by systemically administering to a subject havingcancer in an effective amount to treat the cancer an isolated antibodyor antibody fragment that selectively binds to ABCB5 and achemotherapeutic agent.

The invention in other aspects is an isolated peptide of animmunoglobulin heavy chain variable domain, wherein: (i) CDR14-H1comprises an amino acid sequence of SEQ ID NO. 3; (ii) CDR2-H2 comprisesan amino acid sequence of SEQ ID NO. 4; and (iii) a CDR3-H3 sequence,wherein the isolated peptide is not mAb 3C2-1D12. In some embodimentsthe CDR3-H3 has an amino acid sequence of SEQ ID NO. 3. The isolatedpeptide may bind to human ABCB5 and may be an antibody. Optionally theisolated peptide further includes a light chain variable domain whereinCDR1-L1 has an amino acid sequence of SEQ ID NO. 6, a CDR2-L2 that hasan amino acid sequence of SEQ ID NO. 7 and/or a CDR3-L3 that has anamino acid sequence of SEQ ID NO. 8.

In other aspects the invention is an isolated peptide having animmunoglobulin light chain variable domain, wherein: (i) CDR1-L1 has anamino acid sequence of SEQ ID NO. 6; (ii) CDR2-L2 has an amino acidsequence of SEQ ID NO. 7; and (iii) a CDR3-L3 sequence, wherein theisolated peptide is not mAb 3C2-1D12. In some embodiments the CDR3-L3has an amino acid sequence of SEQ ID NO. 8.

An isolated peptide having at least two antibody variable domains: (a) aheavy chain antibody variable domain comprising the isolated peptide asdescribed herein and (b) a light chain antibody variable domaincomprising the isolated peptide as described herein is providedaccording to other aspects of the invention. In some embodiments theisolated peptide is a single chain Fv. In other embodiments the isolatedpeptide is a Fab isolated peptide. In yet other embodiments the isolatedpeptide is a fully human isolated peptide.

The isolated peptide may further include framework regions FR1, FR2,FR3, and/or FR4 for an isolated peptide variable domain corresponding tothe variant CDR1-H1, CDR2-H2, CDR3-H3, wherein the framework regions areobtained from a single polypeptide template. Each of the frameworkregions may have an amino acid sequence corresponding to the frameworkregion amino acid sequences of polypeptide SEQ ID NO: 1.

In some embodiments the isolated peptide further includes a dimerizationdomain linked to the C-terminal region of a heavy chain polypeptidevariable domain. The dimerization domain may be a leucine zipper domainor a sequence having at least one cysteine residue. The dimerizationdomain has a hinge region in some embodiments. In other embodiments thedimerization domain is a single cysteine. In some embodiments theisolated peptide is a monoclonal antibody. In other embodiments it is abispecific antibody. In yet other embodiments the isolated peptide is asynthetic antibody.

According to another aspect of the invention an anti-ABCB5 antibody orantigen-binding fragment thereof is provided. The antibody has a humanconstant region, wherein the anti-ABCB5 antibody or antigen bindingfragment competitively inhibits binding of mAb 3C2-1D12 to ABCB5. Insome embodiments the antigen-binding fragment is selected from the groupconsisting of Fab, Fab′, F(ab′)₂, Fv, scFv, dsFv, Fd, VH dAb, and VLdAb. In other embodiments the antibody or antigen-binding fragment is ofimmunoglobulin class IgA, IgGb 1, IgG2, IgG3, IgG4 or IgM. In yet otherembodiments the antibody or antigen-binding fragment comprises a humanconstant region and a human variable framework region or theantigen-binding fragment is a single chain antibody. The single chainantibody optionally is a camelid antibody.

A humanized antibody variable domain having a functional antigen bindingregion is provided according to other aspects of the invention. Thehumanized antibody variable domain has non-human CDR1-H1, CDR2-H2,CDR3-H3, CDR1-L1, CDR2-L2, and CDR3-L3 having at least 90% homology toCDR1-H1, CDR2-H2, CDR3-H3, CDR1-L1, CDR2-L2, and CDR3-L3 of mAb 3C2-1D12incorporated into a human antibody variable domain.

In other aspects of the invention a chimeric antibody is provided. Thechimeric antibody has a variable domain which specifically binds toABCB5 and a constant domain, wherein the variable domain and theconstant domain are from different species.

In some embodiments the isolated peptide has an amino acid sequence of aABCB5-binding CDR3-H3 or functionally equivalent variant thereof. Inother embodiments the isolated peptide has an amino acid sequence of aABCB5-binding CDR2-H2 or functionally equivalent variant thereof. Inother embodiments the isolated peptide has an amino acid sequence of aABCB5-binding CDR1-H1 or functionally equivalent variant thereof. Inother embodiments the isolated peptide has an amino acid sequence of aABCB5-binding CDR3-L3 or functionally equivalent variant thereof. Inother embodiments the isolated peptide has an amino acid sequence of aABCB5-binding CDR2-L2 or functionally equivalent variant thereof. In yetother embodiments the isolated peptide has an amino acid sequence of aABCB5-binding CDR1-L1 or functionally equivalent variant thereof.

In other embodiments the isolated peptide is an isolated antibody orantibody fragment. The isolated antibody or antibody fragment mayoptionally be an intact soluble monoclonal antibody. In otherembodiments the isolated antibody or antibody fragment is an isolatedmonoclonal antibody fragment selected from the group consisting of anFab, Fab′, F(ab)₂, Fv, scFv, dsFv, Fd, VH dAb, and VL dAb. In yet otherembodiments the isolated antibody or antibody fragment enhanceschemosensitization. In a preferred embodiment the isolated peptideselectively binds to ABCB5. In yet other embodiments the isolatedantibody or antibody fragment is a humanized antibody. The isolatedpeptide optionally may be a scFv. The isolated peptide in otherembodiments is conjugated to a detectable label. The composition mayalso include a pharmaceutically acceptable carrier and optionally is asterile formulation.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a series of images and graphs depicting a melanoma progressiontissue microarray analysis for ABCB5 as well as a characterization ofABCB5⁺ melanoma populations. FIG. 1( a) shows a chart illustrating ananalysis by the Chromavision Automated Cellular Image System, showingsignificant differences in ABCB5 staining intensities for thin and thickmelanocytic nevi, versus thin and thick primary melanomas, versus lymphnode and visceral melanoma metastases (thin or thick nevi vs. thin orthick primary melanomas, or vs. lymph node or visceral metastases, all Pvalues<0.001; thin primary melanomas vs. thick primary melanomasP=0.004; thin and thick primary melanomas vs. lymph node metastases,P=0.001, lymph node metastases vs. visceral metastases, P=0.025). FIGS.1( b-c) depict several characterizations of ABCB5⁺ melanoma populations.FIG. 1( b) depicts a single-color flow cytometry analysis of clinicalmelanoma samples for expression of ABCB5, CD20, Nestin, TIE-1,VE-cadherin, CD31, or BMPR1a. Illustrated are % positive cells for n=6melanoma patients (horizontal bars indicate mean expression). FIG. 1( c)shows the expression of CD20, Nestin, TIE-1, VE-cadherin, CD31, orBMPR1a by ABCB5⁺ or ABCB5⁻ clinical melanoma cells as determined bydual-color flow cytometry. % positive cells (mean±SEM) are illustratedfor n=3-6 melanoma patients.

FIG. 2 is a series of graphs and images depicting the in vivotumorigenicity of ABCB5⁺ melanoma cell subsets in human to mouse tumorxenograft models. FIG. 2( a) (Left panel) is a graph demonstrating thein vivo tumor formation capacity (%) of unsegregated (US), ABCB5″, orABCB5⁺G3361 melanoma cells following s.c. xenotransplantation (10⁷, 10⁶,or 10⁵ cells/inoculum) into NOD/SCID mice. (Center Panel) is a graphshowing the % inocula without tumor formation plotted against inoculatedcell numbers for unsegregated (US), ABCB5⁻, or ABCB5⁺ G3361 melanomacells into NOD/SCID mice, for determination of the Tumor FormationCapacity 50% (TF₅₀). (Right panel) shows the tumor volumes (mean±SEM) ofprimary melanoma xenografts 8 weeks after s.c. xenotransplantation intoNOD/SCID mice of unsegregated (US), ABCB5⁻, or ABCB5⁺ G3361 melanomacells (10⁷/inoculum). FIG. 2( b) (Left panel) is a graph showing the invivo tumor formation capacity (%) of unsegregated (US), ABCB5⁻, orABCB5⁺ A375 melanoma cells following s.c. xenotransplantation (2×10⁶,2×10⁵, or 2×10⁴ cells/inoculum) into NOD/SCID mice. (Center Panel)depicts the % inocula without tumor formation plotted against inoculatedcell numbers for unsegregated (US), ABCB5″, or ABCB5⁺A375 melanoma cellsinto NOD/SCID mice, for determination of the Tumor Formation Capacity50% (TF₅₀). (Right panel) shows the tumor volumes (mean±SEM) of primarymelanoma xenografts 5 weeks after s.c. xenotransplantation into NOD/SCIDmice of unsegregated (US), ABCB5⁻, or ABCB5⁺ A375 melanoma cells(2×10⁶/inoculum). FIG. 2( c) (Left panel) shows the immunohistochemistryfor ABCB5 expression in a representative primary, unsegregated melanomacell-derived xenograft in NOD/SCID mice, illustrating three discretezones demarcated by dotted lines: ABCB5⁻/melanin-negative (upper left ofpanel), ABCB5⁻/melanin-positive (upper right of panel), andABCB5⁺/melanin-negative (bottom half of panel). (Right panel) is aseries of images of immunofluorescence double staining of frozenmelanoma xenograft sections for coexpression of ABCB5 (FITC) andVE-cadherin (Texas Red). Nuclei are visualized by staining with4′,6-diamidino-2-phenylindole (DAPI, blue). FIG. 2( d) is a graphdepicting the secondary tumor formation capacity (%) in NOD/SCID mice ofABCB5⁻ or ABCB5⁺ cells (10⁷/inoculum) isolated from ABCB5⁺ melanomacell-derived primary tumors. FIG. 2( e) contains two graphs depictingthe in vivo tumor formation capacity (%) (left panel) and tumor volumes(mean±SEM, right panel) of unsegregated (US), ABCB5⁻ or ABCB5 freshlypatient-derived melanoma cells (10⁶/inoculum) 8 weeks after s.c.xenotransplantation into NOD/SCID mice.

FIG. 3 depicts the in vivo tracking of tumorigenicity, self-renewal anddifferentiation of human ABCB5⁺ melanoma cells in NOD/SCID mouserecipients. FIG. 3( a) (Left panels) shows the dual-color flow cytometry(F11 (EYFP) vs. F12 (DsRed2) dot plots) of a tumor cell inoculumconsisting of 10% ABCB5⁺ G3361/DsRed2 and 90% ABCB5⁻ G3361/EYFP cellsprior to xenotransplantation (shown in large panel). Controls (shown insmall panels) are non-transfected G3361 human melanoma cells (top),G3361/DsRed2 cells (middle), and G3361/EYFP cells (bottom). (Rightpanels) show the dual-color flow cytometry (EYFP) vs. F12 (DsRed2) dotplots) of a dissociated xenograft tumor formed 6 weeks after inoculationof 10% ABCB5⁺G3361/DsRed2 and 90% ABCB5⁻ G3361/EYFP cells (shown inlarge panel). Controls (shown in small panels) are non-transfected G3361human melanoma cells (top), G3361/DsRed2 cells (middle), G3361/EYFPcells (bottom). FIG. 3( b) is a graph of the mean percentage (mean±SEM)of DsRed2⁺ cells (% DsRed2⁺/(% DsRed2⁺+% EYFP⁺)×100) of ABCB5⁺ origin orof EYFP⁺ cells (% EYFP⁺/(% DsRed2⁺+% EYFP⁺)×100) of ABCB5⁻ originplotted against weeks post melanoma cell inoculation for resultant invivo tumors at t=4 or 6 weeks (n=3 replicates, respectively) andrespective xenografted cell inocula (n=6). FIG. 3( c) is a series ofdual-channel fluorescence microscopy images of G3361/DsRed2 andG3361/EYFP cells (top and center rows) and of a frozen tissue section(bottom row) derived from in vivo-formed tumors 6 weeks after s.c.xenotransplantation into NOD/SCID mice of 10% G3361/DsRed2 ABCB5⁺ and90% G3361/EYFP ABCB5⁻ cell inocula. The left panels show brightfield,the middle left panels show DsRed2 (ABCB5⁺ origin), the middle rightpanels show EYFP (ABCB5⁻ origin), and the right-most panels show mergedimages (size bars: 25 μm). FIG. 3( d) (Left panels) Depict the flowcytometric analysis of DsRed2 and EYFP expression in ABCB5⁺ cells (top)and ABCB5⁻ cells (bottom) derived from tumors formed in NOD/SCID mice 6weeks after inoculation of 10% ABCB5⁺ G3361/DsRed2 and 90% ABCB5⁻G3361/EYFP cells. (Right panel) is a graph depicting the mean percentage(mean±SD) of either DsRed2 or EYFP fluorescent cells (calculated as %DsRed2⁺/(% DsRed2⁺+% EYFP⁺)×100 or % EYFP⁺/(% DsRed2⁺+% EYFP⁺)×100,respectively) in ABCB5⁺ and ABCB5⁻ cell subsets derived from n=3replicate tumors.

FIG. 4 is a series of graphs and images representing the analysis of theABCB5 mAb effect on melanoma xenograft growth. FIG. 4( a) is a graphmeasuring the tumor volumes (mean±SEM) of melanoma xenografts plottedagainst days after s.c. melanoma cell inoculation into Balb/c nude mice(10⁷ cells/inoculum) for untreated (n=18), isotype control mAb-treated(n=10), or anti-ABCB5 mAb-treated (n=11) animals. [Days of i.p. mAbadministration are indicated by arrows.] FIG. 4( b) is a graph measuringthe tumor formation rate (%) 58 days after s.c. melanoma cellinoculation into Balb/c nude mice (10⁷ cells/inoculum) in untreated(n=18), isotype control mAb-treated (n=10), or anti-ABCB5 mAb-treated(n=11) animals. FIG. 4( c) depicts the ABCB5 immunohistochemistry (leftpanel) and conventional histology (H&E) (right panel) of human melanomaxenografts in nude mice. (Panels represent adjacent sections.) ABCB5⁺regions segregate with unmelanized areas (to left of central dottedline), whereas ABCB5⁻ regions correlate with regions showing particulatebrown-black melanization (to right of central dotted line). FIG. 4( d)shows the flow cytometry analysis (FITC, F11) for surface-bound antibodyin melanoma xenografts, 1 day post i.p. administration of anti-ABCB5 mAb(solid line) or isotype control mAb (shaded). A representative melanomaxenograft isolated from an anti-ABCB5 mAb-treated mouse exhibited 20.5%positivity compared to one derived from an isotype control-treatedanimal. FIG. 4( e) summarizes an assessment of antibody-dependentcell-mediated cytotoxicity (ADCC) by dual color flow cytometry inanti-ABCB5 mAb-, or isotype control mAb-treated or untreated DiO-labeledmelanoma target cell cultures counterstained with propidium iodide (PI)following 24 h coculture with unlabeled effector immune cells derivedfrom Balb/c nude mouse spleens (1:40 target to effector ratios). (Leftpanels) are a series of representative dual-color flow cytometricresults of ADCC with lysed, DIO⁺PI⁺ target cells found in the rightupper quadrants of anti-ABCB5 mAb-treated (top), isotype controlmAb-treated (center), or Ab-untreated (bottom) target/effectorcocultures. (Right panel) depicts an analysis of ADCC (% mean±SEM) inn=6 replicate experiments in treatment groups as above is illustrated([ADCC (%)=(DIO⁺PI⁺ percent sample positivity)−(mean Ab-untreatedDIO⁺PI⁺ percent sample positivity)]).

FIG. 5 summarizes the characterization of unsegregated, ABCB5⁺, orABCB5⁻ human melanoma cells prior to xenotransplantation. FIG. 5( a)depicts the representative flow cytometric surface ABCB5 expression orcontrol staining (FITC, F11) plotted against forward scatter (FSC)determined in unsegregated cultures of human A375 melanoma cells. FIG.5( b) depicts the representative single-color flow cytometric analysisof cell viability for unsegregated (left panels), ABCB5⁺ (center panels)and ABCB5⁻ (right panels) human melanoma cells as determined by cellularincorporation and enzymatic activation of the fluorescent dyecalcein-AM. The upper panels depict calcein-AM samples, the lower panelsno calcein-AM controls. Viable cells are found in the R1 gates of theFSC vs. F12 plots. Figure (c) is a graph showing the ABCB5 expression ofunsegregated and purified ABCB5⁺ or ABCB5⁺-depleted (ABCB5) G3361 humanmelanoma cells.

FIG. 6 is a graph summarizing the correlation analysis of relative ABCB5gene expression with melanoma cell culture doubling times. Pearsoncorrelation of relative ABCB5 gene expression determined by real-timeRT-PCR (mean±SD, n=3 independent experiments) and culture doubling timesof 10 melanoma cell lines (1, LOX IMVI; 2, SK-MEL-5; 3, M14; 4, A375; 5,G3361; 6, UACC-62; 7, SK-MEL-28; 8, UACC-257; 9, SK-MEL-2; 10,MALME-3M); r is the Pearson correlation coefficient.

FIG. 7 is a gel depicting cDNA bands that were produced from the RNAheavy chain (HC) and light chain (LC) variable regions (VRs) byreverse-transcription. Both HC and LC VR PCR products were cloned intothe Invitrogen sequencing vector pCR2.1 and transformed into TOP10cells.

FIG. 8 is 3C2-1D12 antibody HC VR amino acid sequence.

FIG. 9 is 3C2-1D12 antibody HC VR nucleotide sequence.

FIG. 10 is 3C2-1D12 antibody LC VR amino acid sequence.

FIG. 11 is 3C2-1D12 antibody LC VR nucleotide sequence.

FIG. 12 is 3C2-1D12 antibody full length heavy chain nucleotidesequence.

FIG. 13 is 3C2-1D12 antibody full length light chain nucleotidesequence.

DETAILED DESCRIPTION

Tumor initiating cells capable of self-renewal and differentiation,which are responsible for tumor growth, have been identified in humanhematological malignancies and solid cancers. If such minoritypopulations are associated with tumor progression in human patients,specific targeting of tumor initiating cells might provide for a novelstrategy to eradicate cancers currently resistant to systemic therapy. Asubpopulation enriched for human malignant cancer initiating cellsdefined by expression of the chemoresistance mediator ABCB5 have beenidentified according to the invention. As shown in the Examples below,specific targeting of this tumorigenic minority population abrogatestumor growth.

The inventors recently cloned and characterized ABCB5, a novel humanmultidrug resistance transporter shown to be preferentially expressed bycells of melanocytic lineage. Inhibition of ABCB5 renders normallyresistant melanoma cells susceptible to doxorubicin. We havedemonstrated that ABCB5 expression 1) marks tumorigenic melanoma cellsof stem cell phenotype and function; and 2) specific targeting of theABCB5+ melanoma stem cell compartment constitutes a novel, highlypromising stem cell-targeted approach to melanoma therapy. The data isdescribed in more detail in the Examples section.

Additionally, in serial human to mouse xenotransplantation experiments,ABCB5+ melanoma cells possessed greater tumorigenic capacity than ABCB5−bulk populations. Moreover, in vivo genetic cell fate trackingdemonstrated tumorigenic ABCB5+ cancer cells were able to generateABCB5+ and ABCB5− progeny, whereas ABCB5− cells gave rise exclusively toABCB5− progeny. This identification of a specific relationship between achemoresistance mechanism and cancer stem cells in a human malignancyhas important implications for stem cell-targeted approaches to cancertherapy.

It has also been discovered according to the invention that ablation ofABCB5+ melanoma cells via targeted immunotherapeutic approaches mayrepresent a new strategy for achieving more durable clinical responsesthan those obtained by therapeutic strategies directed predominantly atthe bulk population of tumor cells. Therefore, we investigated whetherselective ablation of chemoresistant, tumorigenic human ABCB5+ melanomastem cells via systemic administration of an anti-ABCB5 monoclonalantibody (mAb clone 3C2-1D12) facilitates inhibition of tumorformation/tumor eradication in a relevant preclinical animal model ofhuman malignant melanoma involving human to nude mouse tumor xenografts.

As shown in more detail below, we examined the bioavailability andmelanoma-binding efficacy/specificity of in vivo administered anti-ABCB5mAb in a human to mouse melanoma xenograft model. In order to examinewhether administration of anti-ABCB5 mAb results in detectable in vivoserum levels, mouse sera was incubated with freshly harvested humanmelanoma cell cultures, followed by counterstaining of cells withFITC-conjugated goat anti-mouse Ig secondary Ab and subsequent analysisby single color flow cytometry. Significant binding of FITC-conjugatedgoat anti-mouse Ig secondary Ab to those melanoma cultures pre-incubatedwith sera at all tested dilutions derived from anti-ABCB5 mAb-treatedmice was observed. Binding was not observed with sera derived fromeither isotype control-treated or untreated animals. The detection of5.4% ABCB5 positivity at sera dilutions as low as 1:100 (FIG. 1A) wasconsistent with the previously reported ABCB5+ cell frequency among invitro-cultured G3361 melanoma cells (Frank, N. Y. et al. ABCB5-mediateddoxorubicin transport and chemoresistance in human malignant melanoma.Cancer Res 65, 4320-33 (2005); Frank, N. Y. et al. Regulation ofprogenitor cell fusion by ABCB5 P-glycoprotein, a novel humanATP-binding cassette transporter. J. Biol Chem 278, 47156-65 (2003)).These findings demonstrate that systemically administered anti-ABCB5 mAbresults in effective in vivo mAb serum levels. The data described hereinfurther demonstrate that systemically administered anti-ABCB5 mAbefficiently and preferentially binds xenografted ABCB5+ human melanomacells in vivo, providing evidence for its suitability for in vivotherapeutic targeting approaches. Using human melanoma cells xenograftsinto nude mice, it was demonstrated that specific targeting of theABCB5+ melanoma stem cell compartment with antibodies was an effectivestem cell-targeted approach to melanoma therapy.

The invention is based in part on the discovery, isolation andcharacterization of ABCB5 binding molecules, such as human monoclonalantibodies that bind to ABCB5 and are useful in the treatment of cancer.ABCB5 is a multidrug resistance transporter that is present in cancerousstem cells.

Thus, the compositions of the invention may be useful in the treatmentof a subject having or at risk of having cancer. A subject shall mean ahuman or vertebrate mammal including but not limited to a dog, cat,horse, goat and primate, e.g., monkey. Thus, the invention can also beused to treat diseases or conditions in non human subjects. Forinstance, cancer is one of the leading causes of death in companionanimals (i.e., cats and dogs). Preferably the subject is a human.

As used herein, the term treat, treated, or treating when used withrespect to a disorder such as cancer refers to a prophylactic treatmentwhich increases the resistance of a subject to development of thedisease or, in other words, decreases the likelihood that the subjectwill develop the disease as well as a treatment after the subject hasdeveloped the disease in order to fight the disease, prevent the diseasefrom becoming worse, or slow the progression of the disease compared toin the absence of the therapy.

A subject at risk of developing a cancer is one who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission. Asubject at risk of having cancer also includes a subject havingprecancerous lesions. A precancerous lesion is an area of tissue thathas altered properties and carries the risk of turning into skin cancer.Precancerous lesions may be caused by, for instance, UV radiation,genetics, exposure to carcinogens such as arsenic, tar or x-rayradiation.

A subject having a cancer is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas. Preferably the cancer includes cancer stemcells that express ABCB5.

Optionally, prior to the treatment the presence of ABCB5 positive stemcells can be detected using the binding molecules described herein. Thedetection or diagnosis, methods provided by the invention generallyinvolve contacting one or more molecules of the invention with a samplein or from a subject. Preferably, the sample is first harvested from thesubject, although in vivo detection methods are also envisioned. Thesample may include any body tissue or fluid that is suspected ofharboring the cancer stem cells. For example, the stem cells arecommonly found in or around the tumor mass.

In some aspects, the invention provides binding molecules such aspeptides, antibodies, antibody fragments and small molecules. Themolecules of the invention bind to ABCB5 and enhance tumor killing. Thebinding molecules are referred to herein as isolated molecules thatselectively bind to ABCB5. It is to be understood that such antibodiesare able to bind ABCB5 regardless of its source. Accordingly, antibodiesof the invention that are defined as binding to, for example, melanomacell ABCB5 and capable of detecting and/or enhancing anti-tumor effectsin, for example, melanoma cells as well as other cancers, such as breastcancer.

Although not intending to be bound by any particular theory, it isbelieved that treatment of tumors and cancers may fail becausetumorigenic stem cells are not effectively targeted by conventionaltreatments. The ABCB5 binding molecules of the invention specificallytarget and are involved in the destruction of these cells. Thus, whenthese molecules are used alone or in combination with conventionaltherapies the most aggressive cells of the tumor can be killed.

There are several possible mechanisms by which anti-ABCB5 mAb treatmentmay inhibit in vivo tumorigenic growth and tumor viability of humanmelanoma xenografts in this recipient nude mouse model, includingantibody-dependent cell-mediated cytotoxicity (ADCC),complement-mediated cytotoxicity (CDC) or antibody-dependentmacrophage-mediated cytotoxicity (ABMC), and/or inhibition of ABCB5function, which may contribute to stem cell tumorigenicity. Any of thesemechanisms are predicted to target only the ABCB5-expressing tumor cellsubset compared to controls. We also expect anti-ABCB5 mAb-mediated invivo therapeutic targeting of ABCB5+ melanoma stem cells viachemosensitization- or immunotoxin-mediated cell ablation strategies.Since ABCB5-targeted delivery of toxins (chemical or biological toxins,radionuclides) or of ABCB5 mAb-conjugated siRNAs toward additional tumorstem cell-specific gene targets might require cellular toxininternalization, we also examined cellular internalization of anti-ABCB5mAb following surface binding to ABCB5+ human melanoma cells. Theresults indicate that anti-ABCB5 mAb-conjugated toxins can bespecifically delivered to intracellular compartments in chemoresistantABCB5+ human melanoma cells, highlighting a therapeutic advantage ofthis novel approach for the treatment of clinical melanoma and othercancers.

A molecule that selectively binds to ABCB5 as used herein refers to amolecule, e.g, small molecule, peptide, antibody, fragment, thatinteracts with ABCB5 and optionally interferes with the ABCB5 activity.In some embodiments the molecules are peptides.

The peptides of the invention minimally comprise regions that bind toABCB5. ABCB5-binding regions, in some embodiments derive from theABCB5-binding regions of the antibodies of the invention, oralternatively, they are functionally equivalent variants of suchregions. Accordingly, two particularly important classes ofantibody-derived ABCB5-binding regions are variable regions and CDRs ofthe antibodies described herein. CDR and variable region nucleic acidscan be cloned from antibody-producing cells or prepared syntheticallybased on the sequences described herein.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, antibody fragments, so long as they exhibitthe desired biological activity, and antibody like molecules such asscFv. A native antibody usually refers to heterotetrameric glycoproteinscomposed of two identical light (L) chains and two identical heavy (H)chains. Each heavy and light chain has regularly spaced intrachaindisulfide bridges. Each heavy chain has at one end a variable domain(VH) followed by a number of constant domains. Each light chain has avariable domain at one end (VL) and a constant domain at its other end;the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains.

Certain portions of the variable domains differ extensively in sequenceamong antibodies and are used in the binding and specificity of eachparticular antibody for its particular antigen. However, the variabilityis not evenly distributed throughout the variable domains of antibodies.It is concentrated in three or four segments called“complementarity-determining regions” (CDRs) or “hypervariable regions”in both in the light-chain and the heavy-chain variable domains. Themore highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four or five FR regions, largely adopting a β-sheetconfiguration, connected by the CDRs, which form loops connecting, andin some cases forming part of, the n-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., NIH Publ. No.91-3242, Vol. I, pages 647-669 (1991)). The constant domains are notnecessarily involved directly in binding an antibody to an antigen, butexhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

A hypervariable region or CDR as used herein defines a subregion withinthe variable region of extreme sequence variability of the antibody,which form the antigen-binding site and are the main determinants ofantigen specificity. According to one definition, they can be residues(Kabat nomenclature) 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the lightchain variable region and residues (Kabat nomenclature 31-35 (H1), 50-65(H2), 95-102 (H3) in the heavy chain variable region. Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institute of Health, Bethesda, Md. [1991]).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H1), C_(H2) and C_(H3). The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof. Preferably, the intactantibody has one or more effector functions. Various techniques havebeen developed for the production of antibody fragments. Traditionally,these fragments were derived via proteolytic digestion of intactantibodies (see, e.g., Morimoto et al., Journal of Biochemical andBiophysical Methods 24:107-117 (1992); and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from antibody phage libraries. Alternatively, Fab′-SH fragmentscan be directly recovered from E. coli and chemically coupled to formF(ab′)₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)).According to another approach, F(ab′)₂ fragments can be isolateddirectly from recombinant host cell culture.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain which may be generated by papain digestion ofan intact antibody. The Fc region may be a native sequence Fc region ora variant Fc region. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue at aboutposition Cys226, or from about position Pro230, to the carboxyl-terminusof the Fc region. The Fc region of an immunoglobulin generally comprisestwo constant domains, a CH2 domain and a CH3 domain, and optionallycomprises a CH4 domain. By “Fc region chain” herein is meant one of thetwo polypeptide chains of an Fc region.

The “hinge region,” and variations thereof, as used herein, includes themeaning known in the art, which is illustrated in, for example, Janewayet al., Immuno Biology: the immune system in health and disease,(Elsevier Science Ltd., NY) (4th ed., 1999)

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (K) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Preferably, the ABCB5-binding peptides minimally encompass at least oneCDR from those described herein or those that can be derived from thesequences described herein. As used herein, an ABCB5-binding CDR is aCDR described herein. The ABCB5-binding region may be an ABCB5-bindingCDR1, an ABCB5-binding CDR2, or an ABCB5-binding CDR3, all of which arederived from the antibodies and antibody variable chains disclosedherein.

As used herein, an “ABCB5-binding CDR1” is a CDR1 that binds, preferablyspecifically, to ABCB5, and is derived from either the heavy or lightchain variable regions of the antibodies described herein. It may havean amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO: 6. An “ABCB5-binding CDR2” is a CDR2 that binds,preferably specifically, to ABCB5, and is derived from either the heavyor light chain variable regions of the antibodies described herein. Itmay have an amino acid sequence selected from the group consisting ofSEQ ID NO: 4 and SEQ ID NO: 7. An “ABCB5-binding CDR3” is a CDR3 thatbinds, preferably specifically, to ABCB5, and is derived from either theheavy or light chain variable regions of the antibodies describedherein. It may have an amino acid sequence selected from the groupconsisting of SEQ ID NO: 5 and SEQ ID NO: 8.

In addition to the sequences listed herein, the invention intends toembrace functionally equivalent variants of these sequences includingconservative substitution variants in either the amino acid ornucleotide sequence, as described in greater detail below.

The peptides of the invention are useful inter alia in diagnosticmethods aimed at detecting, in a sample or from a subject, the ABCB5antigen or ABCB5-expressing cells. At a minimum, peptides useful inthese methods need only recognize and bind to ABCB5 regardless ofwhether they also enhance tumor killing. The antibodies may be employed,for instance, in diagnostic FACS analysis, Western blotting, andimmunohistochemistry. Such antibodies may also be employed for in vivodiagnostic uses, where label-conjugated mAbs can be used to assess tumorburden, tumor localization or residual tumor mass following chemotherapyor surgical therapy of ABCB5-expressing tumors. In importantembodiments, the antibodies and fragments thereof bind to ABCB5selectively. In some embodiments, they only possess one or more of theCDRs derived from the antibody clones described herein. In preferredembodiments, the peptides comprise an ABCB5-binding CDR3, and even morepreferably, the peptides comprise a heavy chain ABCB5-binding CDR3. Itis to be understood that not all of the CDRs are required in order toeffect binding to ABCB5. However, in some embodiments the peptidescomprise all of the CDRs of a given antibody clone disclosed herein.

In addition, it should be understood that the invention also embracesthe exchange of CDRs between the variable regions provided herein.Preferably, a heavy chain CDR is exchanged with another heavy chainvariable region CDR, and likewise, a light chain CDR is exchanged withanother light chain variable region CDR.

The peptides may also comprise an ABCB5-binding variable region. AnABCB5-binding variable region is a variable region (preferably anantibody variable region as described herein. SEQ ID NO: 1 correspondsto the amino acid sequences of the heavy chain variable region. SEQ IDNO: 9 corresponds to the nucleotide sequence of the heavy chain variableregion. SEQ ID NO: 2 corresponds to the amino acid sequences of thelight chain variable region. SEQ ID NO: 10 corresponds to the nucleotidesequence of the light chain variable region.

It is to be understood that the nucleic acids or peptides of theinvention may be derived from the sequences provided herein. Thesesequences can be cloned (e.g., by PCR) and inserted into a vector and/orcells in order to produce peptides corresponding to full length variableregions or fragments of full length variable regions, and antibodiescomprising the variable regions. It is therefore possible to generateantibodies or fragments thereof that comprise a combination of light andheavy chain variable regions.

The invention intends to capture antibody and antibody fragments ofvarious isotypes. The antibodies may be of an IgG1, IgG2, IgG3, IgG4,IgD, IgE, IgM, IgA1, IgA2, or sIgA isotype. The invention intends tocapture isotypes found in non-human species as well such as but notlimited to IgY in birds and sharks. Vectors encoding the constantregions of various isotypes are known and previously described. (See,for example, Coloma et al. Novel vectors for the expression of antibodymolecules using variable regions generated by polymerase chain reaction.J Immunol Methods. 1992 Jul. 31; 152(1):89-104; Guttieri et al. Cassettevectors for conversion of Fab fragments into full-length human IgG1monoclonal antibodies by expression in stably transformed insect cells.Hybrid Hybridomics. 2003 June; 22(3):135-45; McLean et al. Human andmurine immunoglobulin expression vector cassettes. Mol. Immunol. 2000October; 37(14):837-45; Walls et al. Vectors for the expression ofPCR-amplified immunoglobulin variable domains with human constantregions. Nucleic Acids Res. 1993 Jun. 25; 21(12):2921-9; Norderhaug etal. Versatile vectors for transient and stable expression of recombinantantibody molecules in mammalian cells. J Immunol Methods. 1997 May 12;204(1):77-87.)

The peptides of the invention are isolated peptides. As used herein, theterm “isolated peptides” means that the peptides are substantially pureand are essentially free of other substances with which they may befound in nature or in vivo systems to an extent practical andappropriate for their intended use. In particular, the peptides aresufficiently pure and are sufficiently free from other biologicalconstituents of their hosts cells so as to be useful in, for example,producing pharmaceutical preparations or sequencing. Because an isolatedpeptide of the invention may be admixed with a pharmaceuticallyacceptable carrier in a pharmaceutical preparation, the peptide maycomprise only a small percentage by weight of the preparation. Thepeptide is nonetheless substantially pure in that it has beensubstantially separated from the substances with which it may beassociated in living systems.

The peptides of the invention bind to ABCB5, preferably in a selectivemanner. As used herein, the terms “selective binding” and “specificbinding” are used interchangeably to refer to the ability of the peptideto bind with greater affinity to ABCB5 and fragments thereof than tonon-ABCB5 derived compounds. That is, peptides that bind selectively toABCB5 will not bind to non-ABCB5 derived compounds to the same extentand with the same affinity as they bind to ABCB5 and fragments thereof,with the exception of cross reactive antigens or molecules made to bemimics of ABCB5 such as peptide mimetics of carbohydrates or variableregions of anti-idiotype antibodies that bind to the ABCB5-bindingpeptides in the same manner as ABCB5. In some embodiments, the peptidesof the invention bind solely to ABCB5 and fragments thereof. As usedherein, a binding peptide that binds selectively or specifically totumor cell ABCB5 may also bind ABCB5 from other sources and will bindwith lesser affinity (if at all) to non-ABCB5 derived compounds. Lesseraffinity may include at least 10% less, 20% less, 30% less, 40% less,50% less, 60% less, 70% less, 80% less, 90% less, or 95% less.

“Isolated antibodies” as used herein refer to antibodies that aresubstantially physically separated from other cellular material (e.g.,separated from cells which produce the antibodies) or from othermaterial that hinders their use either in the diagnostic or therapeuticmethods of the invention. Preferably, the isolated antibodies arepresent in a homogenous population of antibodies (e.g., a population ofmonoclonal antibodies). Compositions of isolated antibodies can howeverbe combined with other components such as but not limited topharmaceutically acceptable carriers, adjuvants, and the like.

“Isolated antibody producing cells” including isolated hybridomas andisolated recombinant cells (such as those described herein), as usedherein, refer to antibody-producing cells that are substantiallyphysically separated from other cells, other bodily material (e.g.,ascites tissue and fluid), and other material that hinders their use inthe production of, for example, an isolated and preferably homogenousantibody population.

Thus in one embodiment, the peptide of the invention is an isolatedintact soluble monoclonal antibody specific for ABCB5. As used herein,the term “monoclonal antibody” refers to a homogenous population ofimmunoglobulins that specifically bind to an identical epitope (i.e.,antigenic determinant). The peptide of the invention in one embodimentis, for example, a monoclonal antibody having a heavy chain variableregion having an amino acid sequence of SEQ ID NO:1 and a light chainvariable region having an amino acid sequence of SEQ ID NO:2. Monoclonalantibodies having any combination of light chain and heavy chainvariable regions are embraced by the invention.

The invention intends to encompass antibodies other than, for example,the sequences of 3C2-1D12, provided that such antibodies have thebinding characteristics of the monoclonal antibodies described herein.Optionally, these additional antibodies also enhance tumor killing ofABCB5-expressing cancer cells. One of ordinary skill in the art caneasily identify antibodies having the functional characteristics of thismonoclonal antibody using the screening and binding assays set forth indetail herein.

Unless indicated otherwise, the term “monoclonal antibody 3C2-1D12” or“mAb3C2-1D12” refers to an antibody that has antigen binding residuesof, or derived from, the murine 3C2-1D12 antibody.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

In other embodiments, the peptide is an antibody fragment. As iswell-known in the art, only a small portion of an antibody molecule, theparatope, is involved in the binding of the antibody to its epitope(see, in general, Clark, W. R. (1986) The Experimental Foundations ofModern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991)Essential Immunology, 7th Ed., Blackwell Scientific Publications,Oxford; and Pier G B, Lyczak J B, Wetzler L M, (eds). Immunology,Infection and Immunity (2004) 1^(st) Ed. American Society forMicrobiology Press, Washington D.C.). The pFc′ and Fc regions of theantibody, for example, are effectors of the complement cascade and canmediate binding to Fc receptors on phagocytic cells, but are notinvolved in antigen binding. An antibody from which the pFc′ region hasbeen enzymatically cleaved, or which has been produced without the pFc′region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. An isolated F(ab′)₂ fragment isreferred to as a bivalent monoclonal fragment because of its two antigenbinding sites. Similarly, an antibody from which the Fc region has beenenzymatically cleaved, or which has been produced without the Fc region,designated an Fab fragment, retains one of the antigen binding sites ofan intact antibody molecule. Proceeding further, Fab fragments consistof a covalently bound antibody light chain and a portion of the antibodyheavy chain denoted Fd (heavy chain variable region). The Fd fragmentsare the major determinant of antibody specificity (a single Fd fragmentmay be associated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

The terms Fab, Fc, pFc′, F(ab′)₂ and Fv are employed with eitherstandard immunological meanings [Klein, Immunology (John Wiley, NewYork, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations ofModern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991)Essential Immunology, 7th Ed., (Blackwell Scientific Publications,Oxford); and Pier G B, Lyczak J B, Wetzler L M, (eds). Immunology,Infection and Immunity (2004) 1^(st) Ed. American Society forMicrobiology Press, Washington D.C.].

In other embodiments, the Fc portions of the antibodies of the inventionmay be replaced so as to produce IgM as well as human IgG antibodiesbearing some or all of the CDRs of the monoclonal antibodies describedherein. Of particular importance is the inclusion of a ABCB5-bindingCDR3 region and, to a lesser extent, the other CDRs and portions of theframework regions of the monoclonal antibodies described herein. Suchhuman antibodies will have particular clinical utility in that they willrecognize and bind, preferably selectively, to ABCB5, but will not evokean immune response in humans against the antibody itself.

The invention also intends to include functionally equivalent variantsof the ABCB5-binding peptides. A “functionally equivalent variant” is acompound having the same function (i.e., the ability to bind to ABCB5)as the peptides of the invention. A functionally equivalent variant maybe peptide in nature but it is not so limited. For example, it may be acarbohydrate, a peptidomimetic, etc. In important embodiments, thefunctionally equivalent variant is a peptide having the amino acidsequence of a variable region or a CDR with conservative substitutionstherein, that is still capable of binding to ABCB5. An example of afunctionally equivalent variant of ABCB5-binding CDR3 from the heavychain variable region (i.e., SEQ ID NO:1) is a peptide havingconservative substitutions in SEQ ID NO:1 which bind, preferablyspecifically, to ABCB5, and optionally which enhances tumor killing ofABCB5-expressing cells.

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. The amino acid sequence variants possess substitutions,deletions, and/or insertions at certain positions within the amino acidsequence of the native amino acid sequence.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart.

Amino acid sequence modification of the antibodies described herein arecontemplated. For example, it may be desirable to improve the bindingaffinity and/or other biological properties of the antibody. Amino acidsequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid alterations may be introduced in the subject antibodyamino acid sequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated.

As used herein, “conservative substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the peptide in which the amino acid substitution ismade. Conservative substitutions of amino acids include substitutionsmade amongst amino acids with the following groups: (1) M,I,L,V; (2)F,Y,W; (3) K,R,H; (4) A,G; (5) S,T; (6) Q,N; and, (7) E,D.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (O)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), H is (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties: (1) hydrophobic: Norleucine, Met,Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues thatinfluence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fe region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g. asubstitution) at one or more amino acid positions including that of ahinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methods ofthe invention may comprise one or more alterations as compared to thewild type counterpart antibody, for e.g. in the Fc region, in additionto the hinge sequence mutation described herein. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Fore.g., it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),for e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants.

Any cysteine residue not involved in maintaining the proper conformationof the anti-ABCB5 antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) may be added to theantibody to improve its stability (particularly where the antibody is anantibody fragment such as an Fv fragment).

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.lycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Functional equivalence refers to an equivalent activity (e.g., bindingto ABCB5, or enhancing killing of ABCB5-expressing cells), however italso embraces variation in the level of such activity. For example, afunctional equivalent is a variant that binds to ABCB5 with lesser,equal, or greater affinity than the monoclonal antibody clones describedherein, provided that the variant is still useful in the invention(i.e., it binds to ABCB5 and optionally enhances tumor killing.

Such substitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding the particular CDR ora peptide comprising the CDR amino acid sequences described herein.These and other methods for altering a CDR containing peptide will beknown to those of ordinary skill in the art and may be found inreferences which compile such methods, e.g. Sambrook or Ausubel, notedabove. In some embodiments, however, due to the size of the CDRs, it maybe more convenient to synthesize the variant peptides using a peptidesynthesizer such as those commercially available. The activity offunctionally equivalent variants of the ABCB5-binding CDR can be testedby the binding assays, and in some cases biological activity assays,discussed in more detail below. As used herein, the terms “functionalvariant”, “functionally equivalent variant” and “functionally activevariant” are used interchangeably.

As used herein the term “functionally active antibody fragment” means afragment of an antibody molecule including an ABCB5-binding region ofthe invention which retains the ability to bind to ABCB5 respectively,preferably in a specific manner. Such fragments can be used both invitro and in vivo. In particular, well-known functionally activeantibody fragments include but are not limited to F(ab)₂, Fab, Fv and Fdfragments of antibodies. These fragments which lack the Fc fragment ofintact antibody, clear more rapidly from the circulation, and may haveless non-specific tissue binding than an intact antibody (Wahl et al.,J. Nucl. Med. 24:316-325 (1983)). As another example, single-chainantibodies can be constructed in accordance with the methods describedin U.S. Pat. No. 4,946,778 to Ladner et al. Such single-chain antibodiesinclude the variable regions of the light and heavy chains joined by aflexible linker moiety. Methods for obtaining a single domain antibody(“Fd”) which comprises an isolated variable heavy chain single domain,also have been reported (see, for example, Ward et al., Nature341:644-646 (1989), disclosing a method of screening to identify anantibody heavy chain variable region (V_(H) single domain antibody) withsufficient affinity for its target epitope to bind thereto in isolatedform). Methods for making recombinant Fv fragments based on knownantibody heavy chain and light chain variable region sequences are knownin the art and have been described, e.g., Moore et al., U.S. Pat. No.4,462,334. Other references describing the use and generation ofantibody fragments include e.g., Fab fragments (Tijssen, Practice andTheory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)), Fv fragments(Hochman et al., Biochemistry 12: 1130 (1973); Sharon et al.,Biochemistry 15: 1591 (1976); Ehrlich et al., U.S. Pat. No. 4,355,023)and portions of antibody molecules (Audilore-Hargreaves, U.S. Pat. No.4,470,925). Thus, those skilled in the art may construct antibodyfragments from various portions of intact antibodies without destroyingthe specificity of the antibodies for ABCB5.

In important aspects of the invention, the functionally active antibodyfragment also retains the ability to enhance killing of ABCB5-expressingcells. In this latter instance, the antibody fragment includes an Fcregion as well as an epitope binding domain. The Fc region allows theantibody fragment to bind to Fc receptor positive cells, whichsubsequently phagocytose the epitope bound by the Fab region of theantibody.

The anti-ABCB5 peptides of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biot,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

An exemplary humanized antibody of interest herein comprises variableheavy domain complementarity determining residues DYYMY (SEQ ID NO:3);TINDGGTHTY (SEQ ID NO:4); and/or DDYYYGSHFDAMDY (SEQ ID NO:5),optionally comprising amino acid modifications of those CDR residues,e.g. where the modifications essentially maintain or improve affinity ofthe antibody. For example, the antibody variant of interest may havefrom about one to about seven or about five amino acid substitutions inthe above variable heavy CDR sequences. Such antibody variants may beprepared by affinity maturation.

The humanized antibody may comprise variable light domaincomplementarity determining residues RASKSVSTSGYSYMH (SEQ ID NO:6);LVSNLES (SEQ ID NO:7); and/or QHIRELTR (SEQ ID NO:8), e.g. in additionto those variable heavy domain CDR residues in the preceding paragraph.Such humanized antibodies optionally comprise amino acid modificationsof the above CDR residues, e.g. where the modifications essentiallymaintain or improve affinity of the antibody. For example, the antibodyvariant of interest may have from about one to about seven or about fiveamino acid substitutions in the above variable light CDR sequences.

The present application also contemplates affinity matured antibodieswhich bind ABCB5. The parent antibody may be a human antibody or ahumanized antibody, e.g., one comprising the variable light and/or heavysequences of SEQ ID Nos. 2 and 1, respectively. The affinity maturedantibody preferably binds to ABCB5 with an affinity superior to that ofmurine mAb3C2-1D12.

Various forms of the humanized antibody or affinity matured antibody arecontemplated. For example, the humanized antibody or affinity maturedantibody may be an antibody fragment, such as a Fab, which is optionallyconjugated with one or more cytotoxic agent(s) in order to generate animmunoconjugate. Alternatively, the humanized antibody or affinitymatured antibody may be an intact antibody, such as an intact IgG1antibody.

European Patent Application 0239400, the entire contents of which ishereby incorporated by reference, provides an exemplary teaching of theproduction and use of humanized monoclonal antibodies in which at leastthe CDR portion of a murine (or other non-human mammal) antibody isincluded in the humanized antibody. Briefly, the following methods areuseful for constructing a humanized CDR monoclonal antibody including atleast a portion of a mouse CDR. A first replicable expression vectorincluding a suitable promoter operably linked to a DNA sequence encodingat least a variable domain of an Ig heavy or light chain and thevariable domain comprising framework regions from a human antibody and aCDR region of a murine antibody is prepared. Optionally a secondreplicable expression vector is prepared which includes a suitablepromoter operably linked to a DNA sequence encoding at least thevariable domain of a complementary human Ig light or heavy chainrespectively. A cell line is then transformed with the vectors.Preferably the cell line is an immortalized mammalian cell line oflymphoid origin, such as a myeloma, hybridoma, trioma, or quadroma cellline, or is a normal lymphoid cell which has been immortalized bytransformation with a virus. The transformed cell line is then culturedunder conditions known to those of skill in the art to produce thehumanized antibody.

As set forth in European Patent Application 0239400 several techniquesare well known in the art for creating the particular antibody domainsto be inserted into the replicable vector. (Preferred vectors andrecombinant techniques are discussed in greater detail below.) Forexample, the DNA sequence encoding the domain may be prepared byoligonucleotide synthesis. Alternatively a synthetic gene lacking theCDR regions in which four framework regions are fused together withsuitable restriction sites at the junctions, such that double strandedsynthetic or restricted subcloned CDR cassettes with sticky ends couldbe ligated at the junctions of the framework regions. Another methodinvolves the preparation of the DNA sequence encoding the variable CDRcontaining domain by oligonucleotide site-directed mutagenesis. Each ofthese methods is well known in the art. Therefore, those skilled in theart may construct humanized antibodies containing a murine CDR regionwithout destroying the specificity of the antibody for its epitope.

As an alternative to humanization, human antibodies can be generated. A“human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any techniques for making human antibodies. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. For example, it is nowpossible to produce transgenic animals (e.g., mice) that are capable,upon immunization, of producing a full repertoire of human antibodies inthe absence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (JH) gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA,90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos.5,591,669, 5,589,369 and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human monoclonal antibodies also may be made by any of the methods knownin the art, such as those disclosed in U.S. Pat. No. 5,567,610, issuedto Borrebaeck et al., U.S. Pat. No. 565,354, issued to Ostberg, U.S.Pat. No. 5,571,893, issued to Baker et al, Kozber, J. Immunol. 133: 3001(1984), Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, p. 51-63 (Marcel Dekker, Inc, new York 1987), and Boerneret al., J. Immunol., 147: 86-95 (1991).

The invention also encompasses the use of single chain variable regionfragments (scFv). Single chain variable region fragments are made bylinking light and/or heavy chain variable regions by using a shortlinking peptide. Any peptide having sufficient flexibility and lengthcan be used as a linker in a scFv. Usually the linker is selected tohave little to no immunogenicity. An example of a linking peptide ismultiple GGGGS residues, which bridge the carboxy terminus of onevariable region and the amino terminus of another variable region. Otherlinker sequences may also be used.

All or any portion of the heavy or light chain can be used in anycombination. Typically, the entire variable regions are included in thescFv. For instance, the light chain variable region can be linked to theheavy chain variable region. Alternatively, a portion of the light chainvariable region can be linked to the heavy chain variable region, orportion thereof. Also contemplated are scFvs in which the heavy chainvariable region is from the antibody of interest, and the light chainvariable region is from another immunoglobulin.

The scFvs can be assembled in any order, for example, V_(H)-linker-V_(L)or V_(L)-linker-V_(H). There may be a difference in the level ofexpression of these two configurations in particular expression systems,in which case one of these forms may be preferred. Tandem scFvs can alsobe made, such as (X)-linker-(X)-linker-(X), in which X are polypeptidesform the antibodies of interest, or combinations of these polypeptideswith other polypeptides. In another embodiment, single chain antibodypolypeptides have no linker polypeptide, or just a short, inflexiblelinker. Possible configurations are V_(L)-V_(H) and V_(H)-V_(L). Thelinkage is too short to permit interaction between V_(L) and V_(H)within the chain, and the chains form homodimers with a V_(L)/V_(H)antigen binding site at each end. Such molecules are referred to in theart as “diabodies”.

Single chain variable regions may be produced either recombinantly orsynthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli, andthe expressed protein may be isolated using standard proteinpurification techniques.

Conditions of expression should be such that the scFv polypeptide canassume optimal tertiary structure. Depending on the plasmid used and thehost cell, it may be necessary to modulate the rate of production. Forinstance, use of a weaker promoter, or expression at lower temperatures,may be necessary to optimize production of properly folded scFv inprokaryotic systems; or it may be preferably to express scFv ineukaryotic cells.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90: 6444-6448 (1993).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity.

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe ABCB5, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit. Methods for makingbispecific antibodies are known in the art. Traditionally, therecombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy-chain/light-chain pairs, wherethe two heavy chains have different specificities [Milstein and Cuello,Nature, 305:537-539 (1983)]. Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

Additionally small peptides including those containing the ABCB5-bindingCDR3 region may easily be synthesized or produced by recombinant meansto produce the peptide of the invention. Such methods are well known tothose of ordinary skill in the art. Peptides can be synthesized, forexample, using automated peptide synthesizers which are commerciallyavailable. The peptides can be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

Peptides, including antibodies, can be tested for their ability to bindto ABCB5 using standard binding assays known in the art. As an exampleof a suitable assay, ABCB5 can be immobilized on a surface (such as in awell of a multi-well plate) and then contacted with a labeled peptide.The amount of peptide that binds to the ABCB5 (and thus becomes itselfimmobilized onto the surface) may then be quantitated to determinewhether a particular peptide binds to ABCB5. Alternatively, the amountof peptide not bound to the surface may also be measured. In a variationof this assay, the peptide can be tested for its ability to binddirectly to a ABCB5-expressing cell.

Peptide binding can also be tested using a competition assay. If thepeptide being tested (including an antibody) competes with themonoclonal antibodies or antibody fragments described herein, as shownby a decrease in binding of the monoclonal antibody or fragment, then itis likely that the peptide and the monoclonal antibody bind to the same,or at least an overlapping, epitope. In this assay system, the antibodyor antibody fragment is labeled and the ABCB5 is immobilized onto thesolid surface. In this way, competing peptides including competingantibodies can be identified. The invention embraces peptides and inparticular antibodies (and fragments thereof) that compete with antibody3C2 1D12 for binding to ABCB5 (i.e., antibodies that recognize and bindto the same epitopes as 3C2 1D12.

The invention also encompasses small molecules that bind to ABCB5 andenhance tumor killing. Such binding molecules may be identified byconventional screening methods, such as phage display procedures (e.g.methods described in Hart et al., J. Biol. Chem. 269:12468 (1994)). Hartet al. report a filamentous phage display library for identifying novelpeptide ligands. In general, phage display libraries using, e.g., M13 orfd phage, are prepared using conventional procedures such as thosedescribed in the foregoing reference. The libraries generally displayinserts containing from 4 to 80 amino acid residues. The insertsoptionally represent a completely degenerate or biased array ofpeptides. Ligands having the appropriate binding properties are obtainedby selecting those phage which express on their surface a ligand thatbinds to the target molecule. These phage are then subjected to severalcycles of reselection to identify the peptide ligand expressing phagethat have the most useful binding characteristics. Typically, phage thatexhibit the best binding characteristics (e.g., highest affinity) arefurther characterized by nucleic acid analysis to identify theparticular amino acid sequences of the peptide expressed on the phagesurface in the optimum length of the express peptide to achieve optimumbinding. Phage-display peptide or antibody library is also described inBrissette R et al Curr Opin Drug Discov Devel. 2006 May; 9(3):363-9.Alternatively, binding molecules can be identified from combinatoriallibraries.

Many types of combinatorial libraries have been described. For instance,U.S. Pat. Nos. 5,712,171 (which describes methods for constructingarrays of synthetic molecular constructs by forming a plurality ofmolecular constructs having the scaffold backbone of the chemicalmolecule and modifying at least one location on the molecule in alogically-ordered array); 5,962,412 (which describes methods for makingpolymers having specific physiochemical properties); and 5,962,736(which describes specific arrayed compounds).

Other binding molecules may be identified by those of skill in the artfollowing the guidance described herein. Library technology can be usedto identify small molecules, including small peptides, which bind toABCB5 and interrupt its function. One advantage of using libraries forantagonist identification is the facile manipulation of millions ofdifferent putative candidates of small size in small reaction volumes(i.e., in synthesis and screening reactions). Another advantage oflibraries is the ability to synthesize antagonists which might nototherwise be attainable using naturally occurring sources, particularlyin the case of non-peptide moieties.

Small molecule libraries can be screened for their modulatory effects onABCB5-mediated rhodamine-123 efflux transport, from which binding toABCB5 can be inferred. Potential substrates or inhibitors of ABCB5function can also be identified by correlating ABCB5 gene or proteinexpression across the NCI-60 panel of cancer cell lines of the NationalCancer Institute with established drug potencies of >100,000 compoundsfor these cell lines, similar as described in Frank et al. CancerResearch 2005 for a select 119 standard anticancer agents.

Many if not all of these compounds can be synthesized using recombinantor chemical libraries. A vast array of candidate compounds can begenerated from libraries of synthetic or natural compounds. Libraries ofnatural compounds in the form of bacterial, fungal, plant and animalextracts are available or can readily produced. Natural andsynthetically produced libraries and compounds can be readily modifiedthrough conventional chemical, physical, and biochemical means. Inaddition, compounds known to bind to and thereby act as antagonists ofcalcium channels may be subjected to directed or random chemicalmodifications such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs which may functionsimilarly or perhaps with greater specificity.

Small molecule combinatorial libraries may also be generated. Acombinatorial library of small organic compounds is a collection ofclosely related analogs that differ from each other in one or morepoints of diversity and are synthesized by organic techniques usingmulti-step processes. Combinatorial libraries include a vast number ofsmall organic compounds. One type of combinatorial library is preparedby means of parallel synthesis methods to produce a compound array. A“compound array” as used herein is a collection of compoundsidentifiable by their spatial addresses in Cartesian coordinates andarranged such that each compound has a common molecular core and one ormore variable structural diversity elements. The compounds in such acompound array are produced in parallel in separate reaction vessels,with each compound identified and tracked by its spatial address.Examples of parallel synthesis mixtures and parallel synthesis methodsare provided in PCT published patent application WO95/18972, publishedJul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and itscorresponding PCT published patent application WO96/22529, which arehereby incorporated by reference.

Standard binding assays are well known in the art, and a number of theseare suitable in the present invention including ELISA, competitionbinding assay (as described above), sandwich assays, radioreceptorassays using radioactively labeled peptides or radiolabeled antibodies,immunoassays, etc. The nature of the assay is not essential provided itis sufficiently sensitive to detect binding of a small number ofpeptides.

A variety of other reagents also can be included in the binding mixture.These include reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalbinding. Such a reagent may also reduce non-specific or backgroundinteractions of the reaction components. Other reagents that improve theefficiency of the assay may also be used. The mixture of the foregoingassay materials is incubated under conditions under which the monoclonalantibody normally specifically binds ABCB5. Such conditions willpreferably mimic physiological conditions. The order of addition ofcomponents, incubation temperature, time of incubation, and otherparameters of the assay may be readily determined. Such experimentationmerely involves optimization of the assay parameters, not thefundamental composition of the assay. Incubation temperatures typicallyare between 4° C. and 40° C. Incubation times preferably are minimizedto facilitate rapid, high throughput screening, and typically arebetween 0.1 and 10 hours. After incubation, the presence or absence ofspecific binding between the peptide and ABCB5 is detected by anyconvenient method available to the user.

Typically, a plurality of assay mixtures are run in parallel withdifferent peptides or different peptide concentrations to obtain adifferent response to the various concentrations. One of theseconcentrations serves as a negative control, i.e., at zero concentrationof ABCB5 or at a concentration of ABCB5 below the limits of assaydetection.

A separation step is often used to separate bound from unbound peptideor antibody. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components (e.g., peptide orantibody) is immobilized on a solid substrate via binding to ABCB5. Theunbound components may be easily separated from the bound fraction. Thesolid substrate can be made of a wide variety of materials and in a widevariety of shapes, e.g., columns or gels of polyacrylamide, agarose orsepharose, microtiter plates, microbeads, resin particles, etc. Theseparation step preferably includes multiple rinses or washes. Forexample, when the solid substrate is a microtiter plate, the wells maybe washed several times with a washing solution, which typicallyincludes those components of the incubation mixture that do notparticipate in specific bindings such as salts, buffer, detergent,non-specific protein, etc. Where the solid substrate is a magnetic bead,the beads may be washed one or more times with a washing solution andisolated using a magnet.

The molecules described herein can be used alone or in conjugates withother molecules such as detection or cytotoxic agents in the detectionand treatment methods of the invention, as described in more detailherein.

Typically, one of the components usually comprises, or is coupled orconjugated to a detectable label. A detectable label is a moiety, thepresence of which can be ascertained directly or indirectly. Generally,detection of the label involves an emission of energy by the label. Thelabel can be detected directly by its ability to emit and/or absorbphotons or other atomic particles of a particular wavelength (e.g.,radioactivity, luminescence, optical or electron density, etc.). A labelcan be detected indirectly by its ability to bind, recruit and, in somecases, cleave another moiety which itself may emit or absorb light of aparticular wavelength (e.g., epitope tag such as the FLAG epitope,enzyme tag such as horseradish peroxidase, etc.). An example of indirectdetection is the use of a first enzyme label which cleaves a substrateinto visible products. The label may be of a chemical, peptide ornucleic acid molecule nature although it is not so limited. Otherdetectable labels include radioactive isotopes such as P³² or H³,luminescent markers such as fluorochromes, optical or electron densitymarkers, etc., or epitope tags such as the FLAG epitope or the HAepitope, biotin, avidin, and enzyme tags such as horseradish peroxidase,β-galactosidase, etc. The label may be bound to a peptide during orfollowing its synthesis. There are many different labels and methods oflabeling known to those of ordinary skill in the art. Examples of thetypes of labels that can be used in the present invention includeenzymes, radioisotopes, fluorescent compounds, colloidal metals,chemiluminescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for thepeptides described herein, or will be able to ascertain such, usingroutine experimentation. Furthermore, the coupling or conjugation ofthese labels to the peptides of the invention can be performed usingstandard techniques common to those of ordinary skill in the art.

Another labeling technique which may result in greater sensitivityconsists of coupling the molecules described herein to low molecularweight haptens. These haptens can then be specifically altered by meansof a second reaction. For example, it is common to use haptens such asbiotin, which reacts with avidin, or dinitrophenol, pyridoxal, orfluorescein, which can react with specific anti-hapten antibodies.

Conjugation of the peptides including antibodies or fragments thereof toa detectable label facilitates, among other things, the use of suchagents in diagnostic assays. Another category of detectable labelsincludes diagnostic and imaging labels (generally referred to as in vivodetectable labels) such as for example magnetic resonance imaging (MRI):Gd(DOTA); for nuclear medicine: ²⁰¹Tl, gamma-emitting radionuclide 99mTc; for positron-emission tomography (PET): positron-emitting isotopes,(18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64,gadodiamide, and radioisotopes of Pb(II) such as 203Pb; 111In.

The conjugations or modifications described herein employ routinechemistry, which chemistry does not form a part of the invention andwhich chemistry is well known to those skilled in the art of chemistry.The use of protecting groups and known linkers such as mono- andhetero-bifunctional linkers are well documented in the literature andwill not be repeated here.

As used herein, “conjugated” means two entities stably bound to oneanother by any physiochemical means. It is important that the nature ofthe attachment is such that it does not impair substantially theeffectiveness of either entity. Keeping these parameters in mind, anycovalent or non-covalent linkage known to those of ordinary skill in theart may be employed. In some embodiments, covalent linkage is preferred.

Noncovalent conjugation includes hydrophobic interactions, ionicinteractions, high affinity interactions such as biotin-avidin andbiotin-streptavidin complexation and other affinity interactions. Suchmeans and methods of attachment are well known to those of ordinaryskill in the art.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

The conjugates of the invention also include an antibody conjugated to acytotoxic agent such as a chemotherapeutic agent, toxin (e.g. anenzymatically active toxin of bacterial, fungal, plant or animal origin,or fragments thereof, or a small molecule toxin), or a radioactiveisotope (i.e., a radioconjugate). Other antitumor agents that can beconjugated to the antibodies of the invention include BCNU,streptozoicin, vincristine and 5-fluorouracil, the family of agentsknown collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).Enzymatically active toxins and fragments thereof which can be used inthe conjugates include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI,PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin and the tricothecenes.

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc⁹⁹m or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc.sup.99m or I.sup.123, .Re.sup.186, Re.sup.188 andIn.sup.111 can be attached via a cysteine residue in the peptide.Yttrium-90 can be attached via a lysine residue. The IODOGEN method(Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can beused to incorporate iodine-123. “Monoclonal Antibodies inImmunoscintigraphy” (Chatal, CRC Press 1989) describes other methods indetail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The sequences responsible for the specificity of the monoclonalantibodies of the invention have been determined. Accordingly, peptidesaccording to the invention can be prepared using recombinant DNAtechnology. There are entities in the United States which will performthis function commercially, such as Thomas Jefferson University and theScripps Protein and Nucleic Acids Core Sequencing Facility (La Jolla,Calif.). For example, the variable region cDNA can be prepared bypolymerase chain reaction using degenerate or non-degenerate primers(derived from the amino acid sequence). The cDNA can be subcloned toproduce sufficient quantities of double stranded DNA for sequencing byconventional sequencing reactions or equipment.

With knowledge of the nucleic acid sequences of the heavy chain andlight chain variable domains of the anti-ABCB5 monoclonal antibody, oneof ordinary skill in the art is able to produce nucleic acids whichencode this antibody or which encode the various antibody fragments,humanized antibodies, or polypeptides described above. It iscontemplated that such nucleic acids will be operably joined to othernucleic acids forming a recombinant vector for cloning or for expressionof the peptides of the invention. The present invention includes anyrecombinant vector containing the coding sequences, or part thereof,whether for prokaryotic or eukaryotic transformation, transfection orgene therapy. Such vectors may be prepared using conventional molecularbiology techniques, known to those with skill in the art, and wouldcomprise DNA coding sequences for the CDR region (and preferably theCDR3 region) and additional variable sequences contributing to thespecificity of the antibodies or parts thereof, as well as othernon-specific peptide sequences and a suitable promoter either with(Whittle et al., Protein Eng. 1:499, 1987 and Burton et al., Science266:1024-1027, 1994) or without (Marasco et al., Proc. Natl. Acad. Sci.(USA) 90:7889, 1993 and Duan et al., Proc. Natl. Acad. Sci. (USA)91:5075-5079, 1994) a signal sequence for export or secretion. Suchvectors may be transformed or transfected into prokaryotic (Huse et al.,Science 246:1275, 1989, Ward et al., Nature 341: 644-646, 1989; Marks etal., J. Mol. Biol. 222:581, 1991 and Barbas et al., Proc. Natl. Acad.Sci. (USA) 88:7978, 991) or eukaryotic (Whittle et al., 1987 and Burtonet al., 1994) cells or used for gene therapy (Marasco et al., 1993 andDuan et al., 1994) by conventional techniques, known to those with skillin the art.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

The expression vectors of the present invention include regulatorysequences operably joined to a nucleotide sequence encoding one of thepeptides of the invention. As used herein, the term “regulatorysequences” means nucleotide sequences which are necessary for, orconducive to, the transcription of a nucleotide sequence which encodes adesired polypeptide and/or which are necessary for or conducive to thetranslation of the resulting transcript into the desired polypeptide.Regulatory sequences include, but are not limited to, 5′ sequences suchas operators, promoters and ribosome binding sequences, and 3′ sequencessuch as polyadenylation signals. The vectors of the invention mayoptionally include 5′ leader or signal sequences, 5′ or 3′ sequencesencoding fusion products to aid in protein purification, and variousmarkers which aid in the identification or selection of transformants.The choice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art. The subsequentpurification of the peptides may be accomplished by any of a variety ofstandard means known in the art.

A preferred vector for screening peptides, but not necessarily preferredfor the mass production of the peptides of the invention, is arecombinant DNA molecule containing a nucleotide sequence that codes forand is capable of expressing a fusion polypeptide containing, in thedirection of amino- to carboxy-terminus, (1) a prokaryotic secretionsignal domain, (2) a polypeptide of the invention, and, optionally, (3)a fusion protein domain. The vector includes DNA regulatory sequencesfor expressing the fusion polypeptide, preferably prokaryotic regulatorysequences. Such vectors can be constructed by those with skill in theart and have been described by Smith et al. (Science 228:1315-1317,1985), Clackson et al. (Nature 352:624-628, 1991); Kang et al. (in“Methods: A Companion to Methods in Enzymology: Vol. 2”, R. A. Lernerand D. R. Burton, ed. Academic Press, NY, pp 111-118,1991); Barbas etal. (Proc. Natl. Acad. Sci. (USA) 88:7978-7982, 1991), Roberts et al.(Proc. Natl. Acad. Sci. (USA) 89:2429-2433, 1992)

A fusion polypeptide may be useful for purification of the peptides ofthe invention. The fusion domain may, for example, include a poly-Histail which allows for purification on Ni+ columns or the maltose bindingprotein of the commercially available vector pMAL (New England BioLabs,Beverly, Mass.). A currently preferred, but by no means necessary,fusion domain is a filamentous phage membrane anchor. This domain isparticularly useful for screening phage display libraries of monoclonalantibodies but may be of less utility for the mass production ofantibodies. The filamentous phage membrane anchor is preferably a domainof the cpIII or cpVIII coat protein capable of associating with thematrix of a filamentous phage particle, thereby incorporating the fusionpolypeptide onto the phage surface, to enable solid phase binding tospecific antigens or epitopes and thereby allow enrichment and selectionof the specific antibodies or fragments encoded by the phagemid vector.

The secretion signal is a leader peptide domain of a protein thattargets the protein membrane of the host cell, such as the periplasmicmembrane of gram negative bacteria. A preferred secretion signal for E.coli is a pelB secretion signal. The predicted amino acid residuesequences of the secretion signal domain from two pelB gene producingvariants from Erwinia carotova are described in Lei, et al. (Nature381:543-546, 1988). The leader sequence of the pelB protein haspreviously been used as a secretion signal for fusion proteins (Better,et al., Science 240:1041-1043, 1988; Sastry, et al., Proc. Natl. Acad.Sci. (USA) 86:5728-5732, 1989; and Mullinax, et al., Proc. Natl. Acad.Sci. (USA) 87:8095-8099, 1990). Amino acid residue sequences for othersecretion signal polypeptide domains from E. coli useful in thisinvention can be found in Oliver, In Neidhard, F.C. (ed.), Escherichiacoli and Salmonella Typhimurium, American Society for Microbiology,Washington, D.C., 1:56-69 (1987).

To achieve high levels of gene expression in E. coli, it is necessary touse not only strong promoters to generate large quantities of mRNA, butalso ribosome binding sites to ensure that the mRNA is efficientlytranslated. In E. coli, the ribosome binding site includes an initiationcodon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotidesupstream from the initiation codon (Shine, et al., Nature 254:34, 1975).The sequence, AGGAGGU, which is called the Shine-Dalgarno (SD) sequence,is complementary to the 3′ end of E. coli 16S rRNA. Binding of theribosome to mRNA and the sequence at the 3′ end of the mRNA can beaffected by several factors: (i) the degree of complementarity betweenthe SD sequence and 3′ end of the 16S rRNA; (ii) the spacing andpossibly the DNA sequence lying between the SD sequence and the AUG(Roberts, et al., Proc. Natl. Acad. Sci. (USA) 76:760, 1979a: Roberts,et al., Proc. Natl. Acad. Sci. (USA) 76:5596, 1979b; Guarente, et al.,Science 209:1428, 1980; and Guarente, et al., Cell 20:543, 1980).Optimization is achieved by measuring the level of expression of genesin plasmids in which this spacing is systematically altered. Comparisonof different mRNAs shows that there are statistically preferredsequences from positions −20 to +13 (where the A of the AUG is position0) (Gold, et al., Annu. Rev. Microbiol. 35:365, 1981). Leader sequenceshave been shown to influence translation dramatically (Roberts, et al.,1979a, b supra); and (iii) the nucleotide sequence following the AUG,which affects ribosome binding (Taniguchi, et al., J. Mol. Biol.,118:533, 1978).

The 3′ regulatory sequences define at least one termination (stop) codonin frame with and operably joined to the heterologous fusionpolypeptide.

In a prokaryotic expression host, the vector utilized includes aprokaryotic origin of replication or replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule extra-chromosomally in a prokaryotic hostcell, such as a bacterial host cell, transformed therewith. Such originsof replication are well known in the art. Preferred origins ofreplication are those that are efficient in the host organism. Aprokaryotic host cell, for instance, is E. coli. For use of a vector inE. coli, a preferred origin of replication is ColE1 found in pBR322 anda variety of other common plasmids. Also preferred is the p15A origin ofreplication found on pACYC and its derivatives. The ColE1 and p15Areplicons have been extensively utilized in molecular biology, areavailable on a variety of plasmids and are described by Sambrook. etal., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold SpringHarbor Laboratory Press, 1989).

In addition, those embodiments that include a prokaryotic repliconpreferably also include a gene whose expression confers a selectiveadvantage, such as drug resistance, to a bacterial host transformedtherewith. Typical bacterial drug resistance genes are those that conferresistance to ampicillin, tetracycline, neomycin/kanamycin orchloramphenicol. Vectors typically also contain convenient restrictionsites for insertion of translatable DNA sequences. Exemplary vectors arethe plasmids pUC18 and pUC19 and derived vectors such as pcDNAIIavailable from Invitrogen (San Diego, Calif.).

When the peptide of the invention is an antibody including both heavychain and light chain sequences, these sequences may be encoded onseparate vectors or, more conveniently, may be expressed by a singlevector. The heavy and light chain may, after translation or aftersecretion, form the heterodimeric structure of natural antibodymolecules. Such a heterodimeric antibody may or may not be stabilized bydisulfide bonds between the heavy and light chains.

A vector for expression of heterodimeric antibodies, such as the intactantibodies of the invention or the F(ab′)₂, Fab or Fv fragmentantibodies of the invention, is a recombinant DNA molecule adapted forreceiving and expressing translatable first and second DNA sequences.That is, a DNA expression vector for expressing a heterodimeric antibodyprovides a system for independently cloning (inserting) the twotranslatable DNA sequences into two separate cassettes present in thevector, to form two separate cistrons for expressing the first andsecond polypeptides of a heterodimeric antibody. The DNA expressionvector for expressing two cistrons is referred to as a dicistronicexpression vector.

Preferably, the vector comprises a first cassette that includes upstreamand downstream DNA regulatory sequences operably joined via a sequenceof nucleotides adapted for directional ligation to an insert DNA. Theupstream translatable sequence preferably encodes the secretion signalas described above. The cassette includes DNA regulatory sequences forexpressing the first antibody polypeptide that is produced when aninsert translatable DNA sequence is directionally inserted into thecassette via the sequence of nucleotides adapted for directionalligation.

The dicistronic expression vector also contains a second cassette forexpressing the second antibody polypeptide. The second cassette includesa second translatable DNA sequence that preferably encodes a secretionsignal, as described above, operably joined at its 3′ terminus via asequence of nucleotides adapted for directional ligation to a downstreamDNA sequence of the vector that typically defines at least one stopcodon in the reading frame of the cassette. The second translatable DNAsequence is operably joined at its 5′ terminus to DNA regulatorysequences forming the 5′ elements. The second cassette is capable, uponinsertion of a translatable DNA sequence (insert DNA), of expressing thesecond fusion polypeptide comprising a secretion signal with apolypeptide coded by the insert DNA.

The peptides of the present invention may also be produced by eukaryoticcells such as CHO cells, human hybridomas, immortalized B-lymphoblastoidcells, and the like. In this case, a vector is constructed in whicheukaryotic regulatory sequences are operably joined to the nucleotidesequences encoding the peptide. The design and selection of anappropriate eukaryotic vector is within the ability and discretion ofone of ordinary skill in the art. The subsequent purification of thepeptides may be accomplished by any of a variety of standard means knownin the art.

In another embodiment, the present invention provides host cells, bothprokaryotic and eukaryotic, transformed or transfected with, andtherefore including, the vectors of the present invention.

Suitable host cells for the expression of glycosylated anti-ABCB5antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

Vertebrate cells are also of particular inteest as host cells. Examplesof useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/−DHFR(CHO, Urlaub et al., Proc.Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HFLA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-ABCB5 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce the anti-ABCB5 antibody of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S.Pat. Re. 30,985 may be used as culture media for the host cells. Any ofthese media may be supplemented as necessary with hormones and/or othergrowth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. The antibodycomposition prepared from the cells can be purified using, for example,hydroxylapatite chromatography, gel electrophoresis, dialysis, andaffinity chromatography, with affinity chromatography being thepreferred purification technique. The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Following anypreliminary purification steps, the mixture may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences, as desired.

The compositions and methods of the invention can be enhanced byutilization in combination with other procedures for cancer andprecancerous lesions. In some instances the treatment procedure involvesadministration of another therapeutic agent such as an anti-canceragent, including but not limited to chemotherapeutic agents andradiation. Chemotherapeutic agents may be selected from the groupconsisting of methotrexate, vincristine, adriamycin, cisplatin, taxol,paclitaxel, non-sugar containing chloroethylnitrosoureas,5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol,fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan,MMI270, BAY 12-9566, RAS farnesyl transferase inhibitor, farnesyltransferase inhibitor, MMP, dacarbazine, LY294002, PX866, MTA/LY231514,LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan,PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin,Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710,VX-853, ZD0101, IS1641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat,CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel,Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine,Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogeneinhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil),Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer, Campto/Levamisole,Camptosar/Irinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine,Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomaldoxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt,ZD1839, LU 79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomaldoxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds,CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide,Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin,PlantinoUcisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel,prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylatingagents such as melphelan and cyclophosphamide, Aminoglutethimide,Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl,Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolideacetate (LHRH-releasing factor analogue), Lomustine (CCNU),Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna,Mitotane), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl,Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastinesulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin,Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), Pentostatin (2′ deoxycoformycin),Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate, butit is not so limited.

The methods of the invention may be performed with therapies fortreating the cancer such as surgery and radiation. The methods of theinvention may also be performed in combination with a therapeutic thatis an isolated short RNA that directs the sequence-specific degradationof a cancer specific mRNA through a process known as RNA interference(RNAi). In some embodiments the cancer-specific mRNA is ABCB5. Theprocess is known to occur in a wide variety of organisms, includingembryos of mammals and other vertebrates. It has been demonstrated thatdsRNA is processed to RNA segments 21-23 nucleotides (nt) in length, andfurthermore, that they mediate RNA interference in the absence of longerdsRNA. Thus, these 21-23 nt fragments are sequence-specific mediators ofRNA degradation and are referred to herein as siRNA or RNAi. Methods ofthe invention encompass the use of these fragments (or recombinantlyproduced or chemically synthesized oligonucleotides of the same orsimilar nature) to enable the targeting of cancer specific mRNAs fordegradation in mammalian cells useful in the therapeutic applicationsdiscussed herein.

The methods for design of the RNA's that mediate RNAi and the methodsfor transfection of the RNAs into cells and animals is well known in theart and the RNAi molecules are readily commercially available (Verma N.K. et al, J. Clin. Pharm. Ther., 28(5):395-404 (2004), Mello C. C. etal. Nature, 431(7006)338-42 (2004), Dykxhoom D. M. et al., Nat. Rev.Mol. Cell. Biol. 4(6):457-67 (2003) Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK)). TheRNAs are preferably chemically synthesized using appropriately protectedribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.Most conveniently, siRNAs are obtained from commercial RNA oligosynthesis suppliers listed herein. In general, RNAs are not toodifficult to synthesize and are readily provided in a quality suitablefor RNAi. A typical 0.2 μmmol-scale RNA synthesis provides about 1milligram of RNA, which is sufficient for 1000 transfection experimentsusing a 24-well tissue culture plate format.

The cancer specific cDNA specific siRNA is designed preferably byselecting a sequence that is not within 50-100 bp of the start codon andthe termination codon, avoids intron regions, avoids stretches of 4 ormore bases such as AAAA, CCCC, avoids regions with GC content <30%or >60%, avoids repeats and low complex sequence, and it avoids singlenucleotide polymorphism sites. The target sequence may have a GC contentof around 50%. The siRNA targeted sequence may be further evaluatedusing a BLAST homology search to avoid off target effects on other genesor sequences. Negative controls are designed by scrambling targetedsiRNA sequences. The control RNA preferably has the same length andnucleotide composition as the siRNA but has at least 4-5 basesmismatched to the siRNA. The RNA molecules of the present invention cancomprise a 3′ hydroxyl group. The RNA molecules can be single-strandedor double stranded; such molecules can be blunt ended or compriseoverhanging ends (e.g., 5′, 3′) from about 1 to about 6 nucleotides inlength (e.g., pyrimidine nucleotides, purine nucleotides). In order tofurther enhance the stability of the RNA of the present invention, the3′ overhangs can be stabilized against degradation. The RNA can bestabilized by including purine nucleotides, such as adenosine orguanosine nucleotides. Alternatively, substitution of pyrimidinenucleotides by modified analogues, e.g., substitution of uridine 2nucleotide 3′ overhangs by 2′-deoxythymidine is tolerated and does notaffect the efficiency of RNAi. The absence of a 2′ hydroxylsignificantly enhances the nuclease resistance of the overhang in tissueculture medium.

The RNA molecules used in the methods of the present invention can beobtained using a number of techniques known to those of skill in theart. For example, the RNA can be chemically synthesized or recombinantlyproduced using methods known in the art. Such methods are described inU.S. Published Patent Application Nos. US2002-0086356A1 andUS2003-0206884A1 that are hereby incorporated by reference in theirentirety.

The methods described herein are used to identify or obtain RNAmolecules that are useful as sequence-specific mediators of cancerspecific mRNA degradation and, thus, for inhibiting proteins whichcontribute to the functioning of cancer cells. Expression of ABCB5, forexample, can be inhibited in humans in order to prevent the protein frombeing translated and thus preventing its function in vivo.

Any RNA can be used in the methods of the present invention, providedthat it has sufficient homology to the cancer specific gene to mediateRNAi. The RNA for use in the present invention can correspond to theentire cancer specific gene or a portion thereof. There is no upperlimit on the length of the RNA that can be used. For example, the RNAcan range from about 21 base pairs (bp) of the gene to the full lengthof the gene or more. In one embodiment, the RNA used in the methods ofthe present invention is about 1000 bp in length. In another embodiment,the RNA is about 500 bp in length. In yet another embodiment, the RNA isabout 22 bp in length. In certain embodiments the preferred length ofthe RNA of the invention is 21 to 23 nucleotides. The Sequence of ABCB5is known, for instance, see U.S. Pat. No. 6,846,883 (which refers toABCB5 as 7p P-glycoprotein).

The ABCB5 binding molecules of the invention are administered to thesubject in an effective amount for treating cancer. An “effective amountfor treating cancer” is an amount necessary or sufficient to realize adesired biologic effect. For example, an effective amount of a compoundof the invention could be that amount necessary to c (i) kill a cancercell; (ii) inhibit the further growth of the cancer, i.e., arresting orslowing its development; and/or (iii) sensitize a cancer cell to ananti-cancer agent or therapeutic. According to some aspects of theinvention, an effective amount is that amount of a compound of theinvention alone or in combination with a cancer medicament, which whencombined or co-administered or administered alone, results in atherapeutic response to the cancer, either in the prevention or thetreatment of the cancer. The biological effect may be the ameliorationand or absolute elimination of symptoms resulting from the cancer. Inanother embodiment, the biological effect is the complete abrogation ofthe cancer, as evidenced for example, by the absence of a tumor or abiopsy or blood smear which is free of cancer cells.

The effective amount of a compound of the invention in the treatment ofa cancer to or in the reduction of the risk of developing a cancer mayvary depending upon the specific compound used, the mode of delivery ofthe compound, and whether it is used alone or in combination. Theeffective amount for any particular application can also vary dependingon such factors as the cancer being treated, the particular compoundbeing administered, the size of the subject, or the severity of thedisease or condition. One of ordinary skill in the art can empiricallydetermine the effective amount of a particular molecule of the inventionwithout necessitating undue experimentation. Combined with the teachingsprovided herein, by choosing among the various active compounds andweighing factors such as potency, relative bioavailability, patient bodyweight, severity of adverse side-effects and preferred mode ofadministration, an effective prophylactic or therapeutic treatmentregimen can be planned which does not cause substantial toxicity and yetis entirely effective to treat the particular subject.

Subject doses of the compounds described herein typically range fromabout 0.1 to 10,000 mg, more typically from about 1 μg/day to 8000 mg,and most typically from about 10 μg to 100 μg. Stated in terms ofsubject body weight, typical dosages range from about 0.1 μg to 20mg/kg/day, more typically from about 1 to 10 mg/kg/day, and mosttypically from about 1 to 5 mg/kg/day. The absolute amount will dependupon a variety of factors including the concurrent treatment, the numberof doses and the individual patient parameters including age, physicalcondition, size and weight. These are factors well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

Multiple doses of the molecules of the invention are also contemplated.In some instances, when the molecules of the invention are administeredwith a cancer medicament a sub-therapeutic dosage of either themolecules or the cancer medicament, or a sub-therapeutic dosage of both,is used in the treatment of a subject having, or at risk of developing,cancer. When the two classes of drugs are used together, the cancermedicament may be administered in a sub-therapeutic dose to produce adesirable therapeutic result. A “sub-therapeutic dose” as used hereinrefers to a dosage which is less than that dosage which would produce atherapeutic result in the subject if administered in the absence of theother agent. Thus, the sub-therapeutic dose of a cancer medicament isone which would not produce the desired therapeutic result in thesubject in the absence of the administration of the molecules of theinvention. Therapeutic doses of cancer medicaments are well known in thefield of medicine for the treatment of cancer. These dosages have beenextensively described in references such as Remington's PharmaceuticalSciences, 18th ed., 1990; as well as many other medical referencesrelied upon by the medical profession as guidance for the treatment ofcancer. Therapeutic dosages of antibodies have also been described inthe art.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular anti-ABCB5 antibodyselected, the particular condition being treated and the dosage requiredfor therapeutic efficacy. The methods of this invention, generallyspeaking, may be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofprotection without causing clinically unacceptable adverse effects.Preferred modes of administration are parenteral routes. The term“parenteral” includes subcutaneous, intravenous, intramuscular,intraperitoneal, and intrasternal injection, or infusion techniques.Other routes include but are not limited to oral, nasal, dermal,sublingual, and local.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

The compounds of the invention can be administered by any ordinary routefor administering medications. Depending upon the type of cancer to betreated, compounds of the invention may be inhaled, ingested oradministered by systemic routes. Systemic routes include oral andparenteral. Inhaled medications are preferred in some embodimentsbecause of the direct delivery to the lung, particularly in lung cancerpatients. Several types of metered dose inhalers are regularly used foradministration by inhalation. These types of devices include metereddose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI),spacer/holding chambers in combination with MDI, and nebulizers.Preferred routes of administration include but are not limited to oral,parenteral, intramuscular, intranasal, intratracheal, intrathecal,intravenous, inhalation, ocular, vaginal, and rectal. For use intherapy, an effective amount of the compounds of the invention can beadministered to a subject by any mode that delivers the nucleic acid tothe affected organ or tissue. “Administering” the pharmaceuticalcomposition of the present invention may be accomplished by any meansknown to the skilled artisan.

According to the methods of the invention, the peptide may beadministered in a pharmaceutical composition. In general, apharmaceutical composition comprises the peptide of the invention and apharmaceutically-acceptable carrier. Pharmaceutically-acceptablecarriers for peptides, monoclonal antibodies, and antibody fragments arewell-known to those of ordinary skill in the art. As used herein, apharmaceutically-acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients, e.g., the ability of the peptide to bind to ABCB5.

Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other materials which arewell-known in the art. Exemplary pharmaceutically acceptable carriersfor peptides in particular are described in U.S. Pat. No. 5,211,657.Such preparations may routinely contain salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The peptides of the invention may be formulated into preparations insolid, semi-solid, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, depositories, inhalants andinjections, and usual ways for oral, parenteral or surgicaladministration. The invention also embraces pharmaceutical compositionswhich are formulated for local administration, such as by implants.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

When the compounds described herein (including peptide and non-peptidevarieties) are used therapeutically, in certain embodiments a desirableroute of administration may be by pulmonary aerosol. Techniques forpreparing aerosol delivery systems containing compounds are well knownto those of skill in the art. Generally, such systems should utilizecomponents which will not significantly impair the biological propertiesof the peptides (see, for example, Sciarra and Cutie, “Aerosols,” inRemington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;incorporated by reference). Those of skill in the art can readilydetermine the various parameters and conditions for producing aerosolswithout resort to undue experimentation.

The peptides of the invention may be administered directly to a tissue.Preferably, the tissue is one in which the cancer stem cells are found.Alternatively, the tissue is one in which the cancer is likely to arise.Direct tissue administration may be achieved by direct injection. Thepeptides may be administered once, or alternatively they may beadministered in a plurality of administrations. If administered multipletimes, the peptides may be administered via different routes. Forexample, the first (or the first few) administrations may be madedirectly into the affected tissue while later administrations may besystemic.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium to alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. Techniques forpreparing aerosol delivery systems are well known to those of skill inthe art. Generally, such systems should utilize components which willnot significantly impair the biological properties of the active agent(see, for example, Sciarra and Cutie, “Aerosols,” in Remington'sPharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporatedby reference). Those of skill in the art can readily determine thevarious parameters and conditions for producing aerosols without resortto undue experimentation.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

In yet other embodiments, the preferred vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT InternationalApplication No. PCT/US/03307 (Publication No. WO 95/24929, entitled“Polymeric Gene Delivery System”, claiming priority to U.S. patentapplication serial no. 213,668, filed Mar. 15, 1994). PCT/US/0307describes a biocompatible, preferably biodegradable polymeric matrix forcontaining a biological macromolecule. The polymeric matrix may be usedto achieve sustained release of the agent in a subject. In accordancewith one aspect of the instant invention, the agent described herein maybe encapsulated or dispersed within the biocompatible, preferablybiodegradable polymeric matrix disclosed in PCT/US/03307. The polymericmatrix preferably is in the form of a microparticle such as amicrosphere (wherein the agent is dispersed throughout a solid polymericmatrix) or a microcapsule (wherein the agent is stored in the core of apolymeric shell). Other forms of the polymeric matrix for containing theagent include films, coatings, gels, implants, and stents. The size andcomposition of the polymeric matrix device is selected to result infavorable release kinetics in the tissue into which the matrix device isimplanted. The size of the polymeric matrix device further is selectedaccording to the method of delivery which is to be used, typicallyinjection into a tissue or administration of a suspension by aerosolinto the nasal and/or pulmonary areas. The polymeric matrix compositioncan be selected to have both favorable degradation rates and also to beformed of a material which is bioadhesive, to further increase theeffectiveness of transfer when the device is administered to a vascular,pulmonary, or other surface. The matrix composition also can be selectednot to degrade, but rather, to release by diffusion over an extendedperiod of time.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the agents of the invention to the subject. Biodegradablematrices are preferred. Such polymers may be natural or syntheticpolymers. Synthetic polymers are preferred. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

In general, the agents of the invention may be delivered using thebioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the peptide, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the platelet reducing agentis contained in a form within a matrix such as those described in U.S.Pat. Nos. 4,452,775, 4,675,189, and 5,736,152 and (b) diffusionalsystems in which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and5,407,686. In addition, pump-based hardware delivery systems can beused, some of which are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for prophylactic treatment of subjects at risk of developing arecurrent cancer. Long-term release, as used herein, means that theimplant is constructed and arranged to delivery therapeutic levels ofthe active ingredient for at least 30 days, and preferably 60 days.Long-term sustained release implants are well-known to those of ordinaryskill in the art and include some of the release systems describedabove.

Therapeutic formulations of the antibodies may be prepared for storageby mixing an antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not intended to limit thescope of the invention. As will be apparent to one of ordinary skill inthe art, the present invention will find application in a variety ofcompositions and methods.

EXAMPLES

Melanoma cells and culture methods. The G3361 human malignant melanomacell line, derived from a single tumor cell cloned in soft agar, wasprovided by Dr. Emil Frei III (Dana-Farber Cancer Institute, Boston,Mass.), the A375 cell line is commercially available from American TypeCulture Collection (ATCC) (Manassas, Va.). All cell lines were culturedin RPMI 1640 medium supplemented with 10% fetal bovine serum, 6 mmol/LHEPES, 2 mmol/l L-glutamine, and 100 IU/ml penicillin/streptomycin at37° C. and 5% CO₂ in a humidified incubator as previously described. TheG3361/DsRed2 and G3361/EYFP cell lines were generated by stabletransfection of G3361 melanoma cells with either Discosoma sp. redfluorescent protein (DsRed2) or the enhanced yellow-green variant (EYFP)of the Aequorea victoria green fluorescent protein (GFP) in conjunctionwith the simian virus 40 large T-antigen nuclear retention signal(Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. A shortamino acid sequence able to specify nuclear location. Cell 39, 499-509(1984)), using pDsRed2-Nuc or pEYFP-Nuc mammalian expression vectorsalso containing a neomycin resistance cassette (BD Biosciences, PaloAlto, Calif.) and the Lipofectamine 2000 reagent (Invitrogen) aspreviously described. Clonal G3361/DsRed and G3361/EYFP cultures weregenerated from stably transfected cultures by limiting dilution.Clinical melanoma cells (n=6 patients) were freshly derived fromsurgical specimen according to human subjects research protocolsapproved by the IRBs of the University of Würzburg Medical School or theWistar Institute, Philadelphia, Pa.

Antibodies. The specific IgG1κ anti-ABCB5 mAb 3C2-1D12 was used hereinin the expression studies. FITC-conjugated 3C2-1D12 mAb was used toassay purity of sorted ABCB5⁺ and ABCB5⁻ melanoma subsets. Unconjugatedor FITC-conjugated MOPC-31C mouse isotype control mAbs, FITC-conjugatedgoat anti-mouse IgG secondary Ab, phycoerythrin (PE)-conjugatedanti-human CD20, anti-human CD31 and isotype control mAbs were purchasedfrom PharMingen, San Diego, Calif. Allophycocyanin (APC)-conjugated andPE-conjugated secondary mAbs were purchased from eBioscience, San Diego,Calif. Unconjugated anti-human TIE-1, anti-human BMPR1a, PE-conjugatedanti-human VE-cadherin and anti-human Nestin mAbs were from R&D Systems,Minneapolis, Minn. The following antibodies were used for ABCB5, TIE-1and VE-cadherin immunohistochemistry and immunofluorescence staining:mouse anti-ABCB5 mAb (Frank, N. Y. et al. ABCB5-mediated doxorubicintransport and chemoresistance in human malignant melanoma. Cancer Res65, 4320-33 (2005); Frank, N. Y. et al. Regulation of progenitor cellfusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassettetransporter. J Biol Chem 278, 47156-65 (2003)), HRP-conjugated horseanti-mouse IgG secondary Ab (Vector Laboratories, Burlingame, Calif.),FITC-conjugated rabbit anti-mouse IgG secondary Ab (ZYMED Laboratories,San Francisco, Calif.), unconjugated rabbit anti-human VE-cadherin Ab(kindly provided by Cell Signaling Technology, Danvers, Mass.), mousecontrol IgG Abs (DAKO, Carpinteria, Calif.), unconjugated rabbit antihuman TIE-1 mAb (Santa Cruz Biotechnologies, Santa Cruz, Calif.),FITC-conjugated donkey anti-mouse IgG secondary Ab, Texas Red-conjugateddonkey anti-rabbit IgG secondary Ab, Cy3-conjugated donkey anti-rabbitIgG secondary Ab, and rabbit control IgG Ab (all from JacksonImmunoResearch, West Grove, Pa.).

Histopathology and immunohistochemistry. 5 micron-thick melanomacryosections were fixed in −20° C. acetone for 5 minutes. Air-driedsections were incubated with 10 μg/ml ABCB5 mAb at 4° C. overnight; 10μg/ml mouse IgG were used as negative control. Sections were washed withPBS×3 for 5 minutes and incubated with 1:200 peroxidase-conjugated horseanti-mouse IgG Ab for ABCB5 staining. For ABCB5/VE-cadherin orABCB5/TIE-1 fluorescence double labeling, 5 μm melanoma sections werefixed in −20° C. acetone for 5 minutes. Air-dried sections wereincubated with 10 μg/ml ABCB5 mAb and 2.5 μg/ml VE-cadherin or TIE-1 Absat 4° C. overnight; 10 μg/ml mouse IgG and 2.5 μg/ml rabbit IgG wereused as negative controls. Sections were washed with PBS containing0.05% tween 20 for 5 minutes×3 and incubated with a 1:150 dilution ofTexas Red-conjugated or Cy3-conjugated donkey anti-rabbit IgG Ab andFITC-conjugated rabbit anti-mouse IgG Ab for 30 minutes at roomtemperature. After subsequent washings, the sections were mounted withVECTASHIELD mounting medium (Vector Laboratories) and covered bycoverslip. Immunofluorescence reactivity was viewed on an OlympusBX51/52 system microscope coupled to a Cytovision system (AppliedImaging, San Jose, Calif.).

Tissue microarray design and analysis. The Melanocytic Tumor ProgressionTMA is the product of a joint effort of the three Skin SPORES (Harvard,M.D. Anderson, University of Pennsylvania). This array contains 480×0.6mm cores of tumor tissue representing four major diagnostic tumor types:benign nevi, primary cutaneous melanoma, lymph node metastasis andvisceral metastasis. Cases were collected from the Pathology services ofthe three participating institutions. For quality control purposes, twoduplicate cores are chosen at each distinct region. Nevi and primarymelanomas had either one region or three regions of the tissue blocksampled (2 or 6 cores), whereas metastatic tumors had one region sampledfrom each block. Therefore, the 480 cores represent 2 adjacent coresfrom 240 distinct histological regions. This array includes 130 coresfrom 35 nevi, 200 cores from 60 primary melanoma and 150 cores from 75metastatic lesions. Operationally, thin nevi and thin melanomas involvedonly the superficial/papillary dermis, whereas thick nevi and thickmelanomas had grown to involve both papillary and deep (reticular)dermis. This array was constructed in the laboratory of Dr. Mark Rubin(Brigham and Women's Hospital Department of Pathology and Dana FarberCancer Institute, Boston). Histologic sections of the tissue array slidewere baked at 58° C. for 20 minutes and then treated as follows:xylene×2 (1 hour, 10 minutes), 100% ethanol×2 for 2 minutes, 95% ethanolfor 2 minutes, and dH₂O×3 for 2 minutes. Antigen retrieval was performedin 10 mMol citrate buffer, pH 6.0 with boiling in pressure cooker for 10minutes and then cooled to room temperature. After washing with PBS×2for 5 minutes, tissue was blocked with 10% horse serum and 1% BSA in PBSat room temperature for 1 hour then incubated with 5 μg/ml ABCB5 mAb at4° C. overnight. The tissue was then washed with PBS-0.05% tween 20×3for 5 minutes then treated with 3% H₂O₂/PBS for 15 minutes. Afterrinsing in PBS, the sections were incubated with 1:200 biotinylatedhorse anti-mouse IgG Ab at room temperature for 30 minutes, rinsed inPBS-tween×3 for 5 minutes, and incubated with avidin-biotin-horseradishperoxidase complex (Vector Laboratories) for 30 minutes at roomtemperature. Immunoreactivity was detected using NovaRed substrate(Vector Laboratories). The Chromavision Automated Cellular ImagingSystem (ACIS) was used to quantify the immunostaining intensity of ABCB5and mIgGIR on the HTMA 84 tissue microarray. The control slide intensityvalues (background plus intrinsic melanization) were subtracted from theexperimental slide and the difference in the intensity values for eachcore was taken to be the true staining. This graph (see FIG. 1) showswith 95% confidence interval the difference in intensity for eachpathology diagnosis. P values between relevant groups were calculatedusing the independent/samples t test. The number above each error barshows the number of cases within each group.

Flow cytometric analysis of ABCB5 expression. Analysis of coexpressionof ABCB5 with the CD20, CD31, VE-cadherin, or BMPR1a surface markers orthe Nestin or TIE-1 intracellular markers in clinical patient-derivedmelanoma cell suspensions was performed by dual-color flow cytometry asdescribed previously. Clinical melanoma cells were incubated withanti-ABCB5 mAb or isotype control mAb or no Ab followed bycounterstaining with APC-conjugated donkey anti-mouse IgG. Cells werethen fixed in PBS containing 2% Paraformaldehyde (30 min at 4° C.), andsubsequently incubated with PE-conjugated anti-CD20, anti-CD31,anti-VE-cadherin, anti-Nestin or PE-conjugated isotype control mAbs, orunconjugated anti-BMPR1a, anti-TIE-1 or unconjugated isotype controlmAbs followed by counterstaining with PE- or FITC-conjugatedanti-immunoglobulin secondary antibodies. Washing steps with stainingbuffer or 1% saponin permeabilization buffer were performed between eachstep. Dual-color flow cytometry was subsequently done with acquisitionof fluorescence emission at the F11 (FITC) or F12 (PE) and F14 (APC)spectra on a Becton Dickinson FACScan (Becton Dickinson, San Jose,Calif.) as described. Statistical differences in expression levels ofthe above listed markers by ABCB5⁺ and ABCB5⁻ cells were determinedusing the nonparametric Mann-Whitney test. A two-sided P value of P<0.05was considered significant. A375 melanoma cells were analyzed forsurface ABCB5 expression by incubation with anti-ABCB5 mAb or isotypecontrol mAb (10 μg/ml) followed by counterstaining with FITC-conjugatedgoat anti-mouse immunoglobulin secondary antibody and single-color flowcytometry (F11) as described.

Cell isolation. Single cell suspensions were generated from humanmelanoma xenografts upon surgical dissection of tumors from sacrificedBalb/c NOD/SCID or Balb/c nude mice 8 weeks following tumor cellinoculation. Each tumor was cut into small pieces (ca. 1 mm³) and tumorfragments were subsequently incubated in 10 ml sterile PBS containing0.1 g/L calcium chloride and 5 mg/ml Collagenase Serva NB6 (SERVAElectrophoresis GmbH, Heidelberg, Germany) for 3 hours at 37° C. on ashaking platform at 200 rpm to generate single cell suspensions.Subsequently, tumor cells were washed with PBS for excess collagenaseremoval. ABCB5⁺ cells were isolated by positive selection and ABCB5⁻cell populations were generated by removing ABCB5⁺ cells usinganti-ABCB5 mAb labeling and magnetic bead cell sorting as described.Briefly, human G3361 or A375 melanoma cells or single cell suspensionsderived from human melanoma xenografts or clinical melanoma samples werelabeled with anti-ABCB5 mAb (20 μg/ml) for 30 min at 4° C., washed forexcess antibody removal, followed by incubation with secondaryanti-mouse IgG mAb-coated magnetic microbeads (Miltenyi Biotec, Auburn,Calif.) and subsequent dual-passage cell separation in MiniMACSseparation columns (Miltenyi Biotec) according to the manufacturersrecommendations. Purity of ABCB5⁺ cell isolates and ABCB5⁻ human G3361melanoma cells was assayed by flow cytometric analysis ofABCB5-expression (F11) on a FACSCalibur machine (Becton Dickinson,Sunnyvale, Calif.) after incubation with FITC-conjugated anti-ABCB5 mAb,followed by anti-mouse IgG mAb-coated microbead incubation and magneticcell sorting. Statistical differences in ABCB5-expression betweenunsegregated, ABCB5⁺, and ABCB5⁻ human G3361 melanoma cells weredetermined using one-way ANOVA followed by the Bonferroni correction. Atwo-sided P value of P<0.05 was considered statistically significant.

Animals. Balb/c nude mice and Balb/c NOD/SCID mice were purchased fromThe Jackson Laboratory (Bar Harbor, Me.). Mice were maintained inaccordance with the institutional guidelines of Children's HospitalBoston and Harvard Medical School and experiments were performedaccording to approved experimental protocols.

Human melanoma xenotransplantation. Unsegregated, ABCB5⁺, or ABCB5⁻human G3361 (10⁷, 10⁶, or 10⁵/inoculum, respectively), or human A375(2×10⁶, 2×10⁵, or 2×10⁴/inoculum, respectively), or clinicalpatient-derived melanoma cells (10⁶/inoculum, respectively), or ABCB5⁺or ABCB5⁻ cells isolated from ABCB5⁺-derived primary G3361 tumorxenografts (10⁷/inoculum, respectively) were injected s.c. uni- orbilaterally into the flanks of recipient Balb/c NOD/SCID mice. Tumorformation/growth was assayed weekly as a time course, at least up to theendpoint of 8 weeks, unless excessive tumor size requiredprotocol-stipulated euthanasia earlier, by determination of tumor volume(TV) according to the established formula [TV(mm³)=π/6×0.5×length×(width)²]. With respect to tumor formation, micewere considered tumor-negative if no tumor tissue was identified uponnecropsy. Statistically significant differences in primary and secondarytumor formation were assessed using the Fisher's Exact test. Differencesin tumor volumes were determined using one-way ANOVA followed by theBonferroni correction or the Kruskal-Wallis Test followed by Dun'scorrection, with two-tailed P values <0.05 considered significant.

In vivo genetic lineage tracking. ABCB5⁺/DsRed2 and ABCB57EYFP humanG3361 tumor cell populations, generated using magnetic bead cell sortingas above, were reconstituted at relative abundance ratios of 1×10⁶ and9×10⁶ cells, respectively, followed by determination of resultant cellratios in inocula by dual-color flow cytometry (F11 (EYFP) vs. F12(DsRed2) plots) prior to xenotransplantation. G3361/DsRed2 andG3361/EYFP co-cultures were injected s.c. (10⁷ cells/inoculum) into theright flank of recipient Balb/c NOD/SCID mice. At 4 or 6 weeks postxenotransplantation, tumors were harvested and single cell suspensionsor frozen tissue sections prepared as above, for determination ofrelative in vivo abundance of DsRed2⁺ and EYFP⁺ melanoma cells bydual-color flow cytometry or fluorescence microscopy of tumor-derivedsingle cell suspensions (upon attachment in adherent tissue cultureplates), and for analysis of 5 μm frozen tissue sections by fluorescencemicroscopy. In additional experiments, the relative abundance of DsRed2⁺and EYFP⁺ melanoma cells was determined in ABCB5⁺ or ABCB5xenograft-derived cell subsets by dual-color flow cytometry as above andthe percentages of DsRed2⁺ and EYFP⁺ tumor cells were statisticallycompared using the unpaired student t test, with a two-sided P value ofP<0.05 considered statistically significant.

Anti-ABCB5 mAb targeting. Unsegregated human G3361 melanoma cells werexenografted s.c. into recipient Balb/c nude mice (10⁷/inoculum). Animalswere injected i.p. with anti-ABCB5 mAb (clone 3C2-1D12), isotype controlmAb (500 μg/injection) bi-weekly or no Ab starting 24 hrs prior tomelanoma xenotransplantation. Tumor growth was assayed bi-weekly as atime course by determination of tumor volume (TV) as described above.Differences in tumor volumes were determined using nonparametric one-wayANOVA (Kruskal-Wallis Test) followed by Dun's correction for comparisonof the three experimental groups, with two-tailed P values <0.05considered significant. For determination of binding efficacy of in vivoadministered anti-ABCB5 mAb to established human to nude mouse melanomaxenografts, single cell suspensions and frozen sections were generatedfrom melanoma xenografts 24 hours following i.p. administration ofanti-ABCB5 mAb, murine IgG1κ isotype control mAb, or no treatment. Theprepared single cell suspensions were subsequently incubated withFITC-conjugated goat anti-mouse Ig secondary Ab for 30 min at 4° C. andanalyzed by single color flow cytometry as above, and frozen sectionswere incubated with HRP-conjugated horse anti-mouse Ig secondary Ab andanalyzed as above.

Assessment of ADCC and CDC. ADCC or CDC were determined by dual-colorflow cytometry as described previously. Briefly, human G3361 melanomacell suspensions in serum-free Dulbecco's Modified Eagle's Medium (DMEM)(BioWhittaker, Walkersville, Md.) were labeled with3,3′-dioctadecyloxacarbocyanine (DiO) (Invitrogen, Carlsbad, Calif.)according to the manufacturers recommendations. DiO-labeled melanomacells were then plated at a density of 300,000 cells per well inflat-bottomed 6-well culture plates in 3 ml and cultured in standardmedium in a humidified incubator overnight. Thereafter, DiO-labeledmelanoma target cells were pre-incubated in the presence or absence ofanti-ABCB5 or isotype control mAbs (20 μg/ml, respectively) for 30 minat 37° C., 5% CO₂, and subsequently cocultured for additional 24 hoursat 37° C., 5% CO₂ with or without freshly isolated Balb/c nude mouseeffector splenocytes (12×10⁶ cells/well, 1:40 target to effector cellratio) for assessment of ADCC, or in the presence or absence of 5%Balb/c nude mouse serum for determination of CDC. Subsequently cells andtheir supernatants were harvested and analyzed by dual-color flowcytometry on a FACSCalibur machine (Becton Dickinson) immediately uponaddition of 10 μg/ml propidium iodide (PI) (Sigma, Milwaukee, WN), withlysed target cells recognized by a DiO⁺PI⁺ phenotype. ADCC levels forthe three treatment groups were calculated as follows: [ADCC(%)=(DIO⁺PI⁺ percent sample positivity)−(mean Ab-untreated DIO⁺PI⁺percent sample positivity)]. Differences in ADCC levels were determinedusing nonparametric one-way ANOVA (Kruskal-Wallis Test) followed byDun's correction, with two-tailed P values <0.05 considered significant.

Cell viability measurements. Cell viability was measured in tumor cellinocula prior to xenotransplantation using calcein-AM staining. Briefly,1×10⁶ unsegregated, ABCB5⁺, or ABCB5⁻ melanoma cells were incubated withcalcein-AM (Molecular Probes, Eugene, Oreg.) for 30 min at 37° C. and 5%CO₂ to allow for substrate-uptake and enzymatic activation to thefluorescent derivative. Subsequently the cells were washed andfluorescence measurements acquired by flow cytometry at the F12 emissionspectrum on a Becton Dickinson FACScan. Cells exhibiting generation ofthe fluorescent calcein-AM derivative compared to unexposed samples wereconsidered viable. Cell viability was also determined in all samplesusing the trypan blue dye exclusion method.

RNA extraction and real-time quantitative reverse transcription-PCR. RNAextraction from G3361 and A375 human melanoma cells and standard cDNAsynthesis reactions were performed using the SuperScript First-StrandSynthesis System for reverse transcription-PCR (Invitrogen) as describedpreviously. Total RNA prepared from 8 additional melanoma cell lines ofthe NCI-60 panel (LOX IMVI, SK-MEL-5, M14, UACC-62, SK-MEL-28, UACC-257,SK-MEL-2, MALME-3M) maintained at the National Cancer Institute underconditions and with passage numbers as described previously was providedby the NCl/NIH Developmental Therapeutics Program. Real-timequantitative reverse transcription-PCR for relative ABCB5 geneexpression was performed as described previously. ABCB5 expression wasassessed by the ratio of the expression level in the sample against meanexpression in all samples, in n=3 independent experiments. Growth data(culture doubling time) for the 8 human melanoma cell lines from theNCI-60 panel were those obtained by the National Cancer Institute, whichcan be found online(http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html). Growthkinetics for the G3361 and A375 melanoma cell lines were established inour laboratory by cell counting according to the formula: to populationdoubling time (h)=T2−T1/(log₂(cell count_(T2)/cell count_(T1))), whereT2 and T1 represent two distinct time points (h) in the logarithmicculture growth phase. Linear correlation of relative ABCB5 mRNAexpression and culture doubling times (h) was performed and a Pearsoncorrelation coefficient was calculated and the criteria of P<0.05 andr>0.3 or r<−0.3 were used to identify significant correlations asdescribed previously.

Example 1

We first examined the relationship of ABCB5 to clinical malignantmelanoma progression, because of its close association with CD166, amarker of more advanced disease. This was assessed via ABCB5immunohistochemical staining and quantitative image analysis of anestablished melanoma progression tissue microarray (TMA) containing 480patient-derived melanoma tissue cores (0.6 mm), representing four majordiagnostic tumor types: benign melanocytic nevi, primary cutaneousmelanoma, melanoma metastases to lymph nodes, and melanoma metastases toviscera (FIG. 1 a). We found that primary or metastatic melanomasexpressed significantly more ABCB5 than benign melanocytic nevi(P<0.001), thick primary melanomas expressed more ABCB5 than thinprimary melanomas (P=0.004), and melanomas metastatic to lymph nodesexpressed more ABCB5 than primary lesions (P=0.001), identifying ABCB5as a novel molecular marker of neoplastic progression in human malignantmelanoma. Apparent heterogeneity in ABCB5 expression was noted inmetastases, with greater staining in lymph node than visceral metastases(P=0.025).

Example 2

When assayed by flow cytometry in single cell suspensions freshlyderived from a smaller series of surgically dissected clinical melanomas(n=6 patients, Table 1), ABCB5 was also found to be consistentlyexpressed in 6 of 6 specimen, with ABCB5⁺ tumor cell frequency rangingfrom 1.6 to 20.4% (9.2±3.2%, mean±SEM) (FIG. 1 b, Table 1). Furtherphenotypic characterization with respect to antigens associated with amore primitive molecular phenotype revealed significant expression ofCD20 in 3 of 6 specimen (frequency in all samples: 0.3±0.2%, mean±SEM),nestin in 6 of 6 (31.9±7.8%), TIE-1 in 6 of 6 (24.9±6.9%), VE-cadherinin 4 of 6 (0.2±0.1%), BMPR1a in 6 of 6 (1.8±1.0%), and of the stromalmarker CD31 in 5 of 6 specimen (0.8±0.4%) (FIG. 1 b). Preferentialexpression by ABCB5⁺ compared to ABCB5⁻ subpopulations, as previouslyidentified for the stem cell determinant CD133, was hereby demonstratedin those samples expressing the respective markers for nestin (49.4±6.6%vs. 26.6±4.9%, respectively, mean±SEM, P=0.026), TIE-1 (59.4±7.8% vs.23.8±7.5%, P=0.015), VE-cadherin (6.4±1.2% vs. 0.1±0.1%, P=0.029), andBMPR1a (37.0±4.4 vs. 2.0±0.2%, P=0.002), but not for CD20 (0.2±0.2% vs.1.1±0.7%, NS), or CD31 (2.4±1.2% vs. 0.5±0.3%, NS) (FIG. 1 c). In situimmunohistochemistry revealed ABCB5⁺ single cells or clusters to accountfor a minority subpopulation within clinical tumors withpositively-stained cells predominantly correlating with non-melanized,undifferentiated regions or TIE-1 expression, and unreactive zonescorresponding to melanized, more differentiated areas.

Table 1 summarizes the tumor characteristics of six patients with amelanoma site (either a metastasis or primary recurrent). Tumors arequantified by % of ABCB5+ present. Also shown is a summary of theoutcomes (number of mice with tumors) for nine groups of NOD/SCID micewhich were transplanted with replicate (n=2-10) inocula of unsegregated,ABCB5⁺ or ABCB5⁻ human melanoma cells.

TABLE 1 Patient and tumor characteristics Number of transplanted micewith ABCB5⁺ in tumors Patient Melanoma tumor Un- no site (%) segregatedABCB5− ABCB5+ P1 Metastasis 8.5 0/2 0/2 2/2 P2 Metastasis 1.6 1/2 0/22/2 P3 Metastasis 3.2 5/5 1/5 5/5 P4 Metastasis 20.4 N/A N/A N/A P5Metastasis 17.4 N/A N/A N/A P6 Primary 4.2 N/A N/A N/A

Example 3

To determine whether the melanoma cell subset defined by ABCB5 wasenriched for MMIC, we compared the abilities of ABCB5⁺-purified (ABCB5⁺)vs. ABCB5⁺-depleted (ABCB5⁻) melanoma cells to initiate tumor formationin vivo, using either established clonal cutaneous human melanomacultures (G3361: 2-10% ABCB5 positivity; A375: 1-10% positivity, FIG. 5a) or freshly patient-derived melanoma cells (FIG. 1 b, Table 1) inhuman to NOD/SCID mouse tumor xenotransplantation experiments. Groups ofNOD/SCID mice were transplanted with replicate (n=2-10) inocula ofunsegregated, ABCB5⁺ or ABCB5⁻ human melanoma cells over a log-foldrange from cell doses unable to efficiently initiate tumor growth(G3361: 10⁵ cells, A375: 2×10⁴ cells) to doses that consistentlyinitiated tumor formation when ABCB5⁺ cells were used (G3361: 10⁷ cells,A375: 2×10⁶ cells, fresh patient isolates: 10⁶ cells). Cell viabilitydetermined by calcein-AM staining exceeded 90% in all tumor cell inoculaand did not significantly differ among isolates (FIG. 5 b).

Of 22 aggregate mice injected with ABCB5″ G3361 melanoma cells only 1mouse transplanted with the highest cell dose generated a tumor (FIG. 2a, left panel). In contrast, 13 of 20 injected with ABCB5⁺ cells formedtumors (P<0.0001), including all mice injected with the highest celldose (FIG. 2 a, left panel, additional P values for individualdose-specific comparisons provided in figure), indicating >2 log-foldenrichment for MMIC in this cell subset, as determined by comparison ofinocula doses required for 50% tumor formation (TF₅₀) (FIG. 2 a, centerpanel).

Similarly, of 21 aggregate mice injected with ABCB5⁻ A375 melanomacells, only 8 mice developed a tumor, whereas 16 of 22 mice injectedwith ABCB5⁺ cells formed tumors (P<0.05), indicating >1 log-foldenrichment for MMIC among ABCB5⁺ A375 cells (FIG. 2 b, left and centerpanels). ABCB5⁺ cell purification resulted in a 19.8-fold enrichment ofABCB5⁺ cell frequency from 5.0±0.4% in unsegregated cultures to98.8±0.8% (mean±SD, n=3, P<0.001) when assayed in representative samplesusing G3361 melanoma cells, and ABCB5⁺-depletion resulted in a 4.75-foldreduction of ABCB5⁺ cell frequency from 5.0±0.4% to 1.1±0.3% (mean±SD,n=3, P<0.001) (FIG. 5 c). This residual contamination (22% of naturallyoccurring ABCB5⁺ frequency) with ABCB5⁺ cells may account for theobserved tumor formation by ABCB5⁻ inocula at the highest doses, andsuggests potential underestimation of MMIC enrichment among ABCB5⁺populations. Notably, in those cases where tumor formation did occur asa result of ABCB5 cell injection at the highest cell doses, tumors wereconsistently found to be smaller than those resulting from ABCB5⁺xenografts (G3361: Tumor Volume (TV)=15±15 vs. 286±90 mm³, respectively,mean±SEM, P<0.01; A375: TV=239±70 vs. 832±121 mm³, respectively,mean±SEM, P<0.05) (FIGS. 2 a and 2 b).

Melanoma culture xenografts were heterogeneous and comprised ABCB5⁺cells predominantly correlating with non-melanized regions andVE-cadherin expression, and ABCB5⁻ zones corresponding to melanizedareas (FIG. 2 c). ABCB5⁺ cells re-purified from ABCB5⁺-derived primarytumors formed secondary tumors more efficiently than their ABCB5⁻counterparts in 11 of 11 vs. 7 of 12 recipients, respectively (P=0.037)(FIG. 2 d) and re-established primary tumor heterogeneity. Consistentwith the results obtained using clonal melanoma model systems, only 1 of9 recipient mice injected with 10⁶ freshly patient-derived ABCB5⁻melanoma cells developed a tumor, whereas all of 9 recipients of 10⁶ABCB5⁺ melanoma cells formed tumors (P<0.001), with the mean TV smallerin recipients of ABCB5⁻ vs. ABCB5⁺ inocula (TV=2±2 vs. 35±11 mm³,respectively, mean±SEM, P<0.01) (FIG. 2 e, Table 1). Tumors generatedfrom ABCB5⁺ melanoma cells re-established naturally-occurring tumorheterogeneity with respect to ABCB5 expression, as determined byimmunohistochemistry and flow cytometry of dissociated tumor specimen,with ABCB5 positivity ranging from 2 to 8% (results not illustrated).These findings establish that MMIC frequency is markedly enriched in themelanoma minority population defined by ABCB5.

Example 4

To directly examine the relative tumor growth contributions ofco-xenografted ABCB5⁺ and ABCB5″ subpopulations, and to furtherinvestigate ABCB5⁺ self-renewal and differentiation capacity, weisolated ABCB5⁺ or ABCB5⁻ melanoma cells from stably transfected G3361cell line variants expressing either red fluorescent protein (DsRed2) orenhanced yellow-green fluorescent protein (EYFP), respectively, a modelsystem designed in our laboratory to allow in vivo genetic lineagetracking. We found that xenotransplantation of ABCB5⁺ G3361/DsRed2 andABCB5⁻ G3361/EYFP fluorochrome transfectant co-cultures reconstituted at14.0±3.0% and 86.0±3.0% relative abundance (mean±SD, n=6), respectively,to NOD/SCID mice resulted in time-dependent, serially increasingrelative frequencies of DsRed2⁺ tumor cells of ABCB5⁺ origin (linearregression slope 6.4±1.0, P<0.0001) in experimental tumors compared toinoculates, up to a frequency of 51.3±1.4% at the experimental endpointof 6 weeks (mean±SD, n=3, P=0.024) (FIGS. 3 a, 3 b, and 3 c top andbottom panels). These findings establish greater tumorigenicity ofABCB5⁺ vs. co-xenografted ABCB5″ melanoma bulk populations in acompetitive tumor development model. Importantly, these results furtherindicate that tumor initiating cells may in addition drive moredifferentiated, and on their own non-tumorigenic cancer bulk populationsto also, albeit less efficiently, contribute to a growing tumor mass.Experimental tumors also contained DsRed21EYFP double-positive melanomacells (FIG. 3 c center panels), indicating that ABCB5⁺-derived tumorcells, like physiological ABCB5⁺ skin progenitors (Frank, N. Y. et al.Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novelhuman ATP-binding cassette transporter. J Biol Chem 278, 47156-65(2003)), engage in cell fusion with ABCB5⁻ subsets.

Example 5

When ABCB5⁺ melanoma cells were purified from experimental tumorsresulting from co-xenotransplantation of 10% ABCB5⁺ G3361/DsRed2 and 90%ABCB5⁻G3361/EYFP fluorochrome transfectants, we found 92.9±6.4%(mean±SD, n=3) of fluorescent cells to be of DsRed2⁺ phenotype (ABCB5⁺origin) (FIG. 3 d, upper left panel), demonstrating self renewalcapacity of this cell subset. EYFP⁺ cells were not found at significantlevels (7.1±6.4%, mean±SD, n=3) among ABCB5⁺ isolates, and the observedlow frequency was fully accounted for in magnitude by the measuredresidual ABCB5⁺ cell contamination among co-grafted ABCB5⁻ EYFP⁺populations (1.1% of 90% EYFP⁺ cells=0.99% vs. 10% ABCB5⁺ DsRed2⁺ cellsin inocula), indicating that ABCB5⁺ tumor cells arose only from ABCB5⁺inocula and that ABCB5″ cells give rise exclusively to ABCB5⁻ progeny.Moreover, fluorescent ABCB5⁻ tumor cell isolates exhibited 52.5±0.8%(mean±SD, n=3) DsRed2 positivity (ABCB5⁺ origin) and 47.5±0.8% EYFPpositivity (ABCB5⁻ origin) (FIG. 3 d, lower left panel), demonstratingthat ABCB5⁺ melanoma cells possess the capacity to differentiate andgive rise to ABCB5⁻ tumor populations. These findings show the existenceof a tumor hierarchy in which ABCB5⁺ melanoma cells, enriched for MMIC,self-renew and give rise to more differentiated, ABCB5⁻ tumor progeny.

Example 6

In order to mechanistically dissect whether the ABCB5-defined,MMIC-enriched minority population is required for tumorigenicity whenunsegregated tumor bulk populations are xenografted, we examined whetherselective killing of this cell subset can inhibit tumor growth andformation. A prospective molecular marker of tumor initiating cells hasnot been targeted to date for in vivo inhibition of tumor growth. Weadministered a monoclonal antibody (mAb) directed at ABCB5 in a human tonude mouse melanoma xenograft model, because nude, as opposed toNOD/SCID, mice are capable of antibody dependent cellular cytotoxicity(ADCC)-mediated tumor cell killing. Melanoma cells were xenografted s.c.into recipient Balb/c nude mice, the animals were injected i.p. withanti-ABCB5 mAb or control mAb bi-weekly starting 24 hrs prior tomelanoma xenotransplantation, and tumor formation and growth wereserially assessed by TV measurements as a time course. Administration ofanti-ABCB5 mAb resulted in significantly inhibited tumor growth comparedto that determined in control mAb-treated or untreated mice over thecourse of a 58-day observation period (mean TV at the endpoint of 58days for anti-ABCB5 mAb-treated=11 mice, no death during the observationperiod) vs. control mAb-treated (n=10 mice, excluding 1 death during theobservation period) or vs. untreated (n=18 mice, excluding 1 deathduring the observation period): 23±16 vs. 325±78 mm³, P<0.01, or vs.295±94 mm³, P<0.001, mean±SEM, respectively) (FIG. 4 a). ControlmAb-treatment showed no significant difference in tumor growth comparedto no treatment (FIG. 4 a). Anti-ABCB5 mAb-treatment also significantlyinhibited tumor formation assessed at 58 days following melanoma cellxenotransplantation, with tumors detected in only 3 of 11 anti-ABCB5mAb-treated mice, vs. 10 of 10 control mAb-treated mice and 18 of 18untreated control animals (P<0.01 and P<0.001, respectively) (FIG. 4 b).

Example 7

Human melanoma xenografts grown in untreated nude mice, like those inNOD/SCID recipients, display tumor heterogeneity and comprise a minoritypopulation of ABCB5⁺ cells predominantly correlating withundifferentiated, non-melanized regions, and ABCB5⁻ zones correspondingto differentiated, melanized areas (FIG. 4 c). Analysis of in vivobinding efficacy revealed that systemically administered anti-ABCB5 mAb,but not control mAb, bound to a subset of tumor cells in establishedmelanoma xenografts (FIG. 4 d) consistent in magnitude with the ABCB5⁺tumor cell subset (FIG. 4 c), as quantitatively determined inxenograft-derived cell suspensions by flow cytometry (FIG. 4 d), andalso by immunohistochemistry by detection of positively staining cellclusters.

Example 8

To determine the mechanism of anti-ABCB5 mAb-mediated inhibition oftumor formation and growth, the immune effector responses ADCC andcomplement-dependent cytotoxicity (CDC) were assessed by dual-color flowcytometry as previously described. Anti-ABCB5 mAb-treated, controlmAb-treated or untreated melanoma target cultures were labeled with thegreen-fluorescent membrane dye DiO and counterstained withred-fluorescent propidium iodide (PI, to which only lysed cells arepermeable), following co-culture with unlabeled effector immune cells orserum derived from Balb/c nude mouse spleens. Anti-ABCB5 mAb but notisotype control mAb significantly induced ADCC-mediated melanoma targetcell death (2.1±0.4% vs. 0.2±0.2%, respectively, P<0.05) in a melanomasubpopulation comparable in size to the ABCB5-expressing subset (Frank,N. Y. et al. ABCB5-mediated doxorubicin transport and chemoresistance inhuman malignant melanoma. Cancer Res 65, 4320-33 (2005)), as determinedfrom the percentage of DiO/PI double-positive cells (FIG. 4 e). Additionof serum to Ab-treated cultures in the absence of effector cells, oraddition of anti-ABCB5 mAb alone under these experimental conditions didnot induce significant cell death compared to controls (results notillustrated), suggesting CDC or direct toxic mAb effects are not thesignificant causes of tumor inhibition in this experimental system.

The effects of ABCB5 targeting on established human to nude mousemelanoma xenografts (n=13 derived from three distinct patients and n=10derived from established melanoma cultures), was examined in order totest the hypothesis that negative selection for MMIC via ADCC-mediatedABCB5+ cell ablation inhibits tumour growth. Such a result would beobserved in a dynamic in vivo situation if the ABCB5+ melanoma subset iscritical to robust tumourigenesis.

Characterization of ABCB5+ or ABCB5− human melanoma cells used inxenotransplantation experiments was undertaken. In vivo anti-ABCB5 mAbadministration, started 14 days following tumour cell inoculation whenxenografts were established (day 0), abrogated the significant tumourgrowth observed in isotype control mAb-treated or untreated groups overthe course of a 21-day treatment period (P<0.001 and P<0.001,respectively) and significantly inhibited mean tumour volume compared tothat determined in either control mAb-treated or untreated mice (TV foranti-ABCB5 mAb-treated (n=23 mice) vs. control mAb-treated (n=22 mice)or vs. untreated (n=22 mice): 32.7±9.4 vs. 226.6±53.8 mm3, P<0.001, orvs. 165.4±36.9 mm³, respectively, mean±s.e.m., P<0.01). The inhibitoryeffects of ABCB5 mAb were also statistically significant when thesubsets of freshly patient-derived melanoma xenograft tumours wereanalyzed independently, with abrogation of the significant tumour growthobserved in isotype control mAb-treated or untreated groups (P<0.05 andP<0.001, respectively) and significantly inhibited mean TV compared tothat determined in either control mAb-treated or untreated mice(anti-ABCB5 mAb-treated (n=13 mice) vs. control mAb-treated (n=12 mice)or vs. untreated (n=12 mice): 29.6±9.2 vs. 289.2±91.8 mm³, P<0.05, orvs. 222.9±57.5 mm³, respectively, mean±s.e.m., P<0.001). ControlmAb-treatment showed no significant effects on tumour growth or tumourvolume compared to no treatment in any of the groups analyzed. Theanimals were sacrificed following the treatment interval as required bythe applicable experimental animal protocol because of tumour burden anddisease state in the patient-derived tumour control groups (measuredmaximal TV: 971.5 mm³).

Immunohistochemical analysis of anti-ABCB5 mAb-treated patient-derivedmelanoma xenografts revealed only small foci of ABCB5 expression(overall <1% of cells) corresponding to in vivo-bound anti-ABCB5 mAb inan adjacent section. An additional adjacent section stained for CD11bdisclosed macrophage infiltration corresponding with regions ofanti-ABCB5 mAb localization, that frequently bordered zones of cellulardegeneration and necrosis. In contrast, control mAb-treated xenograftsrevealed 10-15% ABCB5-reactive cells, secondary anti-Ig mAb failed tolocalize to the respective regions in an adjacent section but detectedregions of intravascular murine immunoglobulin, and CD11b+ macrophagesfailed to infiltrate the tumour tissue. Similar effects were observed incell line-derived melanoma xenografts, with enhanced tumour necrosis inanti-ABCB5 mAb-treated vs. isotype control mAb-treated animals (30-40%vs. <5% necrotic cells, respectively). These findings further supportthe notion that the ABCB5-defined, MMIC enriched minority population isrequired for tumourigenicity.

Characterization of G3361 melanoma xenografts to Balb/c nude mice wasperformed. ABCB5+ regions segregated with unmelanized areas, whereasABCB5-regions correlate with regions showing particulate brown-blackmelanization. Immunohistochemistry of a melanoma xenograft treated withanti-ABCB5 mAb and stained with anti-ABCB5 mAb, secondary anti-Ig Ab orCD11b mAb revealed consistent results to those described above. As inprimary patient-derived xenografts, immunohistochemical analysis ofadjacent tumour sections revealed that systemically administeredanti-ABCB5 mAb bound to ABCB5+ tumour regions, which also correlatedwith CD11b+ cell infiltration. Rare areas of ABCB5 expression to whichin vivo administered antibody failed to localize and into whichCD11b-positive cells failed to infiltrate were also detected.

Example 9

Sequencing of Antibody 3C1 1D12: Total RNA was extracted from thepellets using Fusion Antibodies Ltd in-house RNA extraction protocol.cDNA was created from the RNA by reverse-transcription with an oligo(dT)primer. PCR reactions using variable domain primers to amplify the heavychain (HC) variable region (VR) and light chain (LC) VR regions of themonoclonal antibody DNA gave bands shown in FIG. 7. Both HC and LC VRPCR products were cloned into the Invitrogen sequencing vector pCR2.1and transformed into TOP10 cells. Positive clones for the heavy andlight chain were picked for sequencing analysis. The following sequenceswere obtained.

1. DNA Sequence of full length HC, including signal sequence(underlined)

SEQ ID NO: 17 ATGGACTTTGGGCTGAGCTTGGTTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGCAACTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAGCTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGTCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCCACCATTAATGATGGCGGTACTCACACCTACTATCCAGACAGTCTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACATCCTGTACCTGCAAATGAGCAGTCTGATGTCTGAGGACACAGCCATGTATTATTGTGCAAGAGATGATTATTACTACGGTAGTCACTTCGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 

2. DNA Sequence of full length LC, including signal sequence(underlined)

SEQ ID NO: 18 ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGAGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGCACCAAGCTGGAAATCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA 

3. DNA Sequence of HC VR, including CRDs (underlined)

SEQ ID NO: 9 GAAGTGCAACTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAGCTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGTCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCCACCATTAATGATGGCGGTACTCACACCTACTATCCAGACAGTCTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACATCCTGTACCTGCAAATGAGCAGTCTGATGTCTGAGGACACAGCCATGTATTATTGTGCAAGAGATGATTATTACTACGGTAGTCACTTCGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 

4. DNA Sequence of LC VR, including CRDs (underlined)

SEQ ID NO: 10 GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGAGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTT ACACGTTCGGAGGGGGGCACCAAGCTGGAAATCAAACGG

5. Amino Acid Sequence of HC VR, including Framework Regions (F1, F2,F3, and F4) and CRDs (CDR-H1, CDR-H2 and CDR-H3) as marked. Theframework and CDR regions are determined according to the Kabatnomenclature (E. A. Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition, 1991, NIH).

SEQ ID NO: 1 HC-F1                          CDR-H1EVQLVESGGDLVKPGGSLKLSCAASGFTFS DYYMY HC-F2          CDR-H2     HC-F3WVRQTPEKRLEWVA TINDGGTHTY YPDSLKGRFTISRDNAKNILYLQ                 CDR-H3         HC-F4MSSLMSEDTAMYYCAR DDYYYGSHFDAMDY WGQGTSVTVSS 

6. Amino Acid Sequence of LC VR, including Framework Regions (F1, F2,F3, and F4) and CRDs (CDR-L1, CDR-L2 and CDR-L3) as marked. Theframework and CDR regions are determined according to the Kabatnomenclature (E. A. Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition, 1991, NIH).

SEQ ID NO: 2 LC-F1                   CDR-L1          LC-F2DIVLTQSPASLAVSLGQRATISY RASKSVSTSGYSYMH WNQQKPGQ        CDR-L2  LC-F3                           PPRLLIY LVSNLES EVPARFSGSGSGDTFTLNIHPVEEEDAATYYC  CDR-L3   LC-F4QHIRELTR SEGGTKLEIKR

7. CDR-H1, CDR-H2 and CDR-H3 Sequences:

CDR-H1: DYYMY SEQ ID NO: 3 CDR-H2: TINDGGTHTY SEQ ID NO: 4CDR-H3: DDYYYGSHFDAMDY SEQ ID NO: 5

8. CDR-L1, CDR-L2 and CDR-L3 Sequences:

CDR-L1: RASKSVSTSGYSYMH SEQ ID NO: 6 CDR-L2: LVSNLES SEQ ID NO: 7CDR-L3: QHIRELTR SEQ ID NO: 8

Example 10 Future Studies

We will study the ability to influence melanoma growth and progressionby employing a) two complementary sources of human melanoma (establishedhuman melanoma cell lines and freshly isolated melanoma cells derivedfrom primary and metastatic human tumors); b) two model systems for thestudy of these cells (subcutaneous screening of tumorigenesis inimmunodeficient mice, and more relevant tumorigenesis as it occurs inauthentic human skin xenografts); and c) two alternative strategies formelanoma stem cell abrogation (chemosensitization via ABCB5 functionalblockade, and stem cell killing via immunotoxin or inhibitory siRNAsdelivered specifically to ABCB5+ stem cell targets).

We will investigate whether ABCB5-targeted melanoma stem cellchemoresistance reversal can also inhibit tumor initiation/progressionin chimeric Rag2−/− mouse/human skin xenografts in vivo.

Tumor-targeted immunotoxins have successfully been constructed byconjugating mAbs directed at tumor site-specific antigens to otherwiseindiscriminately cytotoxic agents such as toxins, radionuclides, andgrowth factors. For the proposed studies we will initially focus onutilizing one such molecule, gelonin, a 29 kDa ribosome-inactivatingplant toxin, because gelonin, when used in immunoconjugates directed atmelanoma-specific antigens, has already been demonstrated to exerttumor-specific cytotoxicity in A375 human melanoma xenograft models alsoemployed in this proposal, indicating that gelonin immunoconjugates areexcellent candidates for clinical development. In other future studies,we also envision to study radionuclide immunoconjugates involving forexample Yttrium, which is known to exerts anti-melanoma effects. Whenusing ABCB5-targeted gelonin immunotoxins as a strategy to selectivelyablate ABCB5+ melanoma xenograft subpopulations in vivo, ABCB5-targetedgelonin immunotoxins will involve gelonin/anti-ABCB5 3C2-1D12 mAb orgelonin/isotype control mAb chemical conjugates synthesized and purifiedas previously described. In addition, due to the potential limitationsof intact mAb immunoconjugates with regard to tumor penetration, we willalso use recombinant anti-ABCB5 3C2-1D12 sFv/gelonin fusion proteins,which will be constructed by fusion of the anti-ABCB5 3C2-1D12 sFv gene,generated as above, to gelonin DNA, using the splice-overlap extensionPCR method. The recombinant fusion immunotoxin will be expressed in E.coli and purified as previously described. Recombinant controlsFv/gelonin fusion proteins will be generated in an identical mannerfrom isotype control mAb-producing murine hybridoma cell lines.

We will also develop and use ABCB5 antibody-mediated, targetcell-specific delivery of siRNAs to specific oncogenes as a strategy toselectively inhibit ABCB5+ melanoma tumor stem populations in vivo.While delivery of small interfering RNAs (siRNAs) into cells has untilrecently been a key obstacle to their in vivo therapeutic application, anovel approach involving antibody/protamine fusion proteins as siRNAdelivery vehicles, has recently demonstrated efficacy in systemic,cell-type specific siRNA delivery to melanoma tumors in experimentalanimal models in vivo, and proved effective in inhibiting in vivomelanoma growth when siRNAs directed at MYC, MDM2 and VEGF wereantibody-targeted to a model receptor expressed on B16 murine melanomacells. This approach takes advantage of the nucleic acid-bindingproperties of protamine, which normally nucleates DNA in sperm, to bindsiRNAs of various specificities and deliver them to cells bearing aspecific cell surface marker when protamine is fused to antibody Fabfragments or sFv specifically directed to such a marker. In order toutilize this strategy to target ABCB5-expressing melanoma stem cells, wewill construct a recombinant anti-ABCB5 3C2-1D12 sFv/protamine fusionprotein (ABCB5 sFv-P), by fusion of the anti-ABCB5 3C2-1D12 sFv gene toprotamine DNA, using the splice-overlap extension PCR method. Therecombinant fusion protein ABCB5 sFv-P will be expressed and purified aspreviously described. ABCB5 sFv-P will initially be used to deliversiRNA targeted to MYC, since gene-targeted MYC down-regulation inhibitsin vivo tumor growth not only in murine B16 melanoma, but also in micebearing established human melanoma xenografts, leading to extensivetumor cell apoptosis via induction of p53 and inhibition of Bcl-2proteins. We have already found MYC consistently expressed in ABCB5+human melanoma subpopulations. Furthermore, gene expression of human MYCcan be effectively inhibited by RNAi approaches, and MYC-targeting siRNAoligonucleotides validated in these studies are commercially availablefrom Dharmacon, Inc. (Chicago, Ill.). In the proposed studies, the ABCB5sFv-P binding capacity for MYC siRNA, the ABCB5 sFv-P-mediated MYC siRNAtarget cell delivery and resultant MYC gene inhibition, and ABCB5sFv-P/MYC siRNA-mediated blockade of tumor cell proliferation will firstbe examined in vitro in human G3361 and A375 melanoma cultures exactlyas described previously.

The in vivo study protocol for ABCB5+ melanoma stem cell targeting willemploy the human to mouse tumor xenograft models utilizing both NOD-SCIDmice as well as chimeric Rag2−/−/human skin chimeric mice as recipientsof human melanoma xenografts either derived from established cell linesor freshly isolated from human patients, exactly as already describedabove. In a first set of experiments aimed at assessing the effects ofimmunotoxins (ABCB5 mAb/gelonin or sFv/gelonin) or of ABCB5 sFv-P/MYCsiRNA on tumor initiation, immunotoxins (ABCB5 mAb/gelonin orsFv/gelonin or controls) will be administered in 0.25 ml sterile PBS viatail vein injection, and ABCB5 sFv-P complexed to MYC siRNA or controlswill be administered on days 0, 1 and 3 after tumor implantation viatail vein injection (80 μg siRNA in an injection volume of 100 μl at amolar ratio of ABCB5 sFv-P/total siRNA of 1:6) to murine recipients ofhuman melanoma cell xenografts randomized on day 0 followingxenotransplantation into the following treatment and control groups(n=10 replicate animals for each melanoma cell line and for each tumorcell specimen freshly isolated from each of n=10 primary melanomas andn=10 melanoma metastases, xenografted s.c. to NOD-SCID mice orintradermally to human skin/Rag2−/− mice chimera): 1) ABCB5 mAb/gelonin500 μg/mouse i.v. q.o.d. starting at day 0; 2) isotype controlmAb/gelonin 500 μg/mouse i.v. q.o.d. starting at day 0; 3) ABCB5sFv/gelonin 500 μg/mouse i.v. q.o.d. starting at day 0; 4) controlsFv/gelonin 500 μg/mouse i.v. q.o.d. starting at day 0; 5) ABCB5sFv-P/MYC siRNA i.v. on days 0, 1 and 3; 6) ABCB5 sFv-P/control siRNAi.v. on days 0, 1 and 3; 7) ABCB5 sFv-P i.v. on days 0, 1 and 3. Thetreatment protocol is summarized in Table 2:

TABLE 2 Group No. mice Treatment 1 10 ABCB5 mAb/gelonin 500 μg/mousei.v. q.o.d. starting at day 0 2 10 isotype control mAb/gelonin 500μg/mouse i.v. q.o.d. starting at day 0 3 10 ABCB5 sFv/gelonin 500μg/mouse i.v. q.o.d. starting at day 0 4 10 control sFv/gelonin 500μg/mouse i.v. q.o.d. starting at day 0 5 10 ABCB5 sFv-P/MYC siRNA i.v.on days 0, 1 and 3 6 10 ABCB5 sFv-P/control siRNA i.v. on days 0, 1 and3 7 10 ABCB5 sFv-P i.v. on days 0, 1 and 3

In a second set of experiments aimed at assessing the effects ofimmunotoxins (ABCB5 mAb/gelonin or sFv/gelonin) or of ABCB5 sFv-P/MYCsiRNA on tumor progression of established tumors, murine recipients ofhuman melanoma cell xenografts will be randomized on day 7 followingxenotransplantation (when tumors are established) into the treatment andcontrol groups summarized in Table 7 (n=10 replicate animals for eachmelanoma cell line and for each tumor cell specimen freshly isolatedfrom each of n=10 primary melanomas and n=10 melanoma metastases,xenografted s.c. to NOD-SCID mice or intradermally to human skin/Rag2−/−mice chimera):

TABLE 3 Group No. mice Treatment 8 10 ABCB5 mAb/gelonin 500 μg/mousei.v. q.o.d. starting at day 7 9 10 isotype control mAb/gelonin 500μg/mouse i.v. q.o.d. starting at day 7 10 10 ABCB5 sFv/gelonin 500μg/mouse i.v. q.o.d. starting at day 7 11 10 control sFv/gelonin 500μg/mouse i.v. q.o.d. starting at day 7 12 10 ABCB5 sFv-P/MYC siRNA i.v.on days 7, 8 and 10 13 10 ABCB5 sFv-P/control siRNA i.v. on days 7, 8and 10 14 10 ABCB5 sFv-P i.v. on days 7, 8 and 10

Clinical tumor formation/growth will be assayed daily as a time courseby determination of tumor volume (TV) according to the establishedformula [TV (mm3)=π/6×0.5×length×(width)2] for the length of theexperiment (45 days). Statistically significant differences in tumorformation as a function of the applied treatment regimen will beassessed using the Fisher's Exact test. Differences in tumor volumesbetween experimental groups will be determined using nonparametricANOVA. Two-tailed P values <0.05 will be considered statisticallysignificant. Immunofluorescent and immunohistochemical analysis of eachtransplanted tumor xenograft dissected from animals of all treatmentgroups sacrificed initially on day 45 of the experiment (sequentialsacrifices [e.g. at days 10, 20, 30, and 45] will be performed based onthe day 45 findings, and in addition to examination of primary tumors,sacrificed animals will be necropsied, all metastases evaluated, and alltissues pathologically evaluated for evidence of toxicity mediated bythe applied treatment regimen). Expression of ABCB5 and co-expression ofABCB5 with CD 133 will be assayed by sequential HRP/AP-immunoenzymaticdouble staining of frozen melanoma xenograft sections as previouslydescribed. Tumor sections will be analyzed by brightfield microscopy,and mean percentages of cells staining positive for each marker will besemiquantitatively (no positivity: −; <10% positivity: +; 10-50%positivity: ++; >50% positivity: +++) classified based on cell countingin three microscopy fields (400× magnification) for each stainingcondition as previously described. Using fluorescent microscopy andseparate filters for each fluorochrome, RFP-positive cells (ABCB5+origin) and GFP-positive cells (ABCB5-origin) will be counted (100cells/sample) and RFP/GFP cell ratios within each tumor will becalculated. Mean ratios derived from replicate animals subjected to eachtreatment regimen will be statistically compared using nonparametricANOVA. To assess efficacy of ABCB5+ targeting strategies, apoptoticmelanoma cells growing in the murine subcutis, human skin xenografts,and at sites of metastasis will be identified according to establishedcriteria used for light microscopy and confirmed by the TUNEL assay. Wewill also screen immunohistochemically for protein expression relevantto apoptotic pathways, including Bax, Bcl-2, and Bcl-XL. Finally theseresults will be correlated with a screen for cell proliferation-relatedmarkers (MIB-1, PCNA, and cyclin D1/D3). Positive cells will beenumerated manually over cross-sectional profiles, and by the use ofcomputer-assisted imaging programs available in the co-PI's laboratory(GFM) that should significantly enhance efficiency of quantitation.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1. A method of delivering a therapeutic agent to an intracellularcompartment of a cell, comprising: contacting a cell with an isolatedmolecule that selectively binds to ABCB5 conjugated to a therapeuticagent in an effective amount to deliver the therapeutic agent to anintracellular compartment of the cell.
 2. The method of claim 1, whereinthe isolated molecule that selectively binds to ABCB5 is an isolatedpeptide.
 3. The method of claim 2, wherein the isolated peptide is anantibody or antigen binding fragment thereof.
 4. The method of claim 1,wherein the therapeutic agent is a toxin.
 5. The method of claim 1,wherein the therapeutic agent is an siRNA.
 6. The method of claim 1,wherein the therapeutic agent is a chemotherapeutic agent.
 7. The methodof claim 1, wherein the therapeutic agent is a therapeutic antibody. 8.The method of claim 1, further comprising contacting a cell with anisolated molecule that selectively binds to a surface marker selectedfrom the group consisting of CD49e, CD133, and CD166.
 9. The method ofclaim 1, further comprising contacting a cell with an isolated moleculethat selectively binds to a surface marker selected from the groupconsisting of BMPR1a, TIR-1, VE-cadherin (CD144) and nestin.
 10. Amethod for treating a subject, comprising systemically administering anisolated peptide that selectively binds to ABCB5 and comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,and SEQ ID NO:8, or functionally equivalent variants thereof containingconservative substitutions to a subject having cancer in an effectiveamount to treat the cancer.
 11. A method for treating a subject,comprising administering a composition comprising an isolated peptidethat selectively binds to ABCB5 and comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ IDNO:8, or functionally equivalent variants thereof containingconservative substitutions, wherein the isolated peptide is not mAb3C2-1D12 to a subject having cancer in an effective amount to treat thecancer.
 12. A method for treating a subject, comprising administering anisolated peptide that selectively binds to ABCB5 and comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7and SEQ ID NO:8, or functionally equivalent variants thereof containingconservative substitutions and a chemotherapeutic agent, to a subjecthaving cancer in an effective amount to treat the cancer.
 13. A methodfor treating a subject, comprising systemically administering anisolated antibody or antibody fragment that selectively binds to ABCB5and a chemotherapeutic agent to a subject having cancer in an effectiveamount to treat the cancer.